Purine derivatives

ABSTRACT

The present invention relates to compounds of formula 1 
                         
or pharmaceutically acceptable salts thereof, wherein
     one of R 1  and R 2  is methyl, ethyl or isopropyl, and the other is H;   R 3  and R 4  are each independently H, branched or unbranched C 1 -C 6  alkyl, or aryl, and wherein at least one of R 3  and R 4  is other than H;   R 5  is a branched or unbranched C 1 -C 5  alkyl group or a C 1 -C 6  cycloalkyl group, each of which may be optionally substituted with one or more OH groups;   R 6 , R 7 , R 8  and R 9  are each independently H, halogen, NO 2 , OH, OMe, CN, NH 2 , COOH, CONH 2 , or SO 2 NH 2 .   
     A further aspect of the invention relates to pharmaceutical compositions comprising compounds of formula 1, and the use of said compounds in treating proliferative disorders, viral disorders, stroke, alopecia, CNS disorders, neurodegenerative disorders, or diabetes.

RELATED APPLICATIONS

This application is a continuation of PCT/GB2003/003554, filed on Aug.13, 2003, which claims priority to GB 0219054.4 filed on Aug. 15, 2002.The entire contents of both of these applications are herebyincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to new 2,6,9-substituted purinederivatives and their biological applications. In particular, theinvention relates to purine derivatives having antiproliferativeproperties which are useful in the treatment of proliferative disorderssuch as cancer, leukemia, psoriasis and the like.

BACKGROUND

Initiation, progression, and completion of the mammalian cell cycle areregulated by various cyclin-dependent kinase (CDK) complexes, which arecritical for cell growth. These complexes comprise at least a catalytic(the CDK itself) and a regulatory (cyclin) subunit. Some of the moreimportant complexes for cell cycle regulation include cyclin A(CDK1—also known as cdc2, and CDK2), cyclin B1-B3 (CDK1), cyclin D1-D3(CDK2, CDK4, CDK5, CDK6), cyclin E (CDK2). Each of these complexes isinvolved in a particular phase of the cell cycle. Not all members of theCDK family are involved exclusively in cell cycle control, however. ThusCDKs 7, 8, and 9 are implicated in the regulation of transcription, andCDK5 plays a role in neuronal and secretory cell function.

The activity of CDKs is regulated post-translationally, by transitoryassociations with other proteins, and by alterations of theirintracellular localisation. Tumour development is closely associatedwith genetic alteration and deregulation of CDKs and their regulators,suggesting that inhibitors of CDKs may be useful anti-cancertherapeutics. Indeed, early results suggest that transformed and normalcells differ in their requirement for e.g. cyclin A/CDK2 and that it maybe possible to develop novel antineoplastic agents devoid of the generalhost toxicity observed with conventional cytotoxic and cytostatic drugs.While inhibition of cell cycle-related CDKs is clearly relevant in e.g.oncology applications, this may not be the case for the inhibition ofRNA polymerase-regulating CDKs. On the other hand, inhibition ofCDK9/cyclin T function was recently linked to prevention of HIVreplication and the discovery of new CDK biology thus continues to openup new therapeutic indications for CDK inhibitors (Sausville, E. A.Trends Molec. Med. 2002, 8,S32-S37).

The function of CDKs is to phosphorylate and thus activate or deactivatecertain proteins, including e.g. retinoblastoma proteins, lamins,histone H1, and components of the mitotic spindle. The catalytic stepmediated by CDKs involves a phospho-transfer reaction from ATP to themacromolecular enzyme substrate. Several groups of compounds (reviewedin e.g. Fischer, P. M. Curr. Opin. Drug Discovery Dev. 2001, 4, 623-634)have been found to possess anti-proliferative properties by virtue ofCDK-specific ATP antagonism.

WO 98/05335 (CV Therapeutics Inc) discloses 2,6,9-trisubstituted purinederivatives that are selective inhibitors of cell cycle kinases. Suchcompounds are useful in the treatment of autoimmune disorders, e.g.rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis;treating cancer, cardiovascular disease, such as restenosis, host vgraft disease, gout, polycystic kidney disease and other proliferativediseases whose pathogenesis involves abnormal cell proliferation.

WO 99/07705 (The Regents of the University of California) disclosespurine analogues that inhibit inter alia protein kinases, G-proteins andpolymerases. More specifically, the invention relates to methods ofusing such purine analogues to treat cellular proliferative disordersand neurodegenerative diseases.

WO 97/20842 (CNRS) also discloses purine derivatives displayingantiproliferative properties which are useful in treating cancer,psoriasis, and neurodegenerative disorders.

The present invention seeks to provide new 2,6,9-substituted purinederivatives, particularly those having antiproliferative properties.

STATEMENT OF INVENTION

A first aspect of the invention relates to a compound of formula I

or a pharmaceutically acceptable salt thereof, whereinone of R¹ and R² is methyl, ethyl or isopropyl, and the other is H;R³ and R⁴ are each independently H, branched or unbranched C₁-C₆ alkyl,or aryl, and wherein at least one of R³ and R⁴ is other than H;R⁵ is a branched or unbranched C₁-C₅ alkyl group or a C₁-C₆ cycloalkylgroup, each of which may be optionally substituted with one or more OHgroups;R⁶, R⁷, R⁸ and R⁹ are each independently H, halogen, NO₂, OH, OMe, CN,NH₂, COOH, CONH₂, or SO₂NH₂.

A second aspect of the invention relates to a pharmaceutical compositioncomprising a compound of formula 1 and a pharmaceutically acceptablecarrier, diluent or excipient.

A third aspect of the invention relates to the use of a compound offormula 1 in the preparation of a medicament for treating one or more ofthe following disorders:

-   -   a proliferative disorder;    -   a viral disorder;    -   a stroke;    -   alopecia;    -   a CNS disorder;    -   a neurodegenerative disorder; and    -   diabetes.

A fourth aspect of the invention relates to the use of a compound offormula 1 as an anti-mitotic agent.

A fifth aspect of the invention relates to the use of a compound offormula 1 for inhibiting a protein kinase.

A sixth aspect of the invention relates to a method of treating aproliferative disease, said method comprising administering to a mammala therapeutically effective amount of a compound of formula 1.

A seventh aspect of the invention relates to the use of a compound ofthe invention in an assay for identifying further candidate compoundsthat influence the activity of one or more CDK enzymes.

DETAILED DESCRIPTION

As mentioned above, a first aspect of the invention relates to acompound of formula 1 as defined hereinbefore.

It is known in the art that the main in vivo metabolic deactivationpathway of the experimental anti-proliferative CDK-inhibitory agentroscovitine (refer PCT Intl. Patent Appl. Publ. WO 97/20842; Wang, S.,McClue, S. J., Ferguson, J. R., Hull, J. D., Stokes, S., Parsons, S.,Westwood, R., and Fischer, P. M. Tetrahedron: Asymmetry 2001, 12,2891-2894) comprises oxidation of the carbinol group to a carboxyl groupand subsequent excretion of this metabolite [Nutley, B. P., Raynaud, F.I., Wilson, S. C., Fischer, P., McClue, S., Goddard, P. M., Jarman, M.,Lane, D., and Workman, P. Clin. Cancer Res. 2000, 6 Suppl. (Proc.11^(th) AACR—NCI-EORTC Intl. Conf. #318)]. Authentic synthetic materialidentical with this metabolite, shows reduced biological activity invitro. Thus, roscovitine and the carboxyl derivative inhibit CDK2/cyclinE activity with IC₅₀ values of 0.08 and 0.24 μM, respectively.Similarly, the average anti-proliferative IC₅₀ values in arepresentative panel of human transformed tumour cell lines forroscovitine and the carboxyl derivative were ca. 10 and >50 μM,respectively.

Thus, in one preferred embodiment, the invention seeks to provide newpurine derivatives which exhibit improved resistance to metabolicdeactivation.

In one preferred embodiment of the invention, one of R¹ and R² is ethylor isopropyl, and the other is H.

In another preferred embodiment of the invention, R⁵ is isopropyl orcyclopentyl.

In one preferred embodiment, R⁶, R⁷, R⁸ and R⁹ are all H.

In one preferred embodiment, R¹ or R² is ethyl and the other is H.

In one preferred embodiment, R³ and R⁴ are each independently H, methyl,ethyl, propyl, butyl or phenyl.

Thus, in one preferred embodiment, R³ and R⁴ are each independently H,methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl or phenyl.

In a more preferred embodiment, R³ and R⁴ are each independently H,methyl, ethyl, propyl or butyl.

Thus, in one preferred embodiment, R³ and R⁴ are each independently H,methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl or t-butyl.

In an even more preferred embodiment, R³ and R⁴ are each independentlyH, methyl, ethyl, isopropyl or t-butyl.

In one especially preferred embodiment, said compound of formula 1 isselected from the following:

-   (2S3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2-ol;-   (2R3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2-ol;-   (3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-3-ol;-   (3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-3-ol;-   (3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-hexan-3-ol;-   (3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-hexan-3-ol;-   (3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol;-   (3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol;-   (3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol;-   (3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol;-   (3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol;    and-   (3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol.

Even more preferably, said compound of formula 1 is selected from thefollowing:

-   (2S3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2-ol;-   (2R3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2-ol;-   (3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-3-ol;-   (3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-3-ol;-   (3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol;-   (3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol;    and-   (3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-3-ylamino}-2-methyl-pentan-2-ol.

More preferably still, said compound of formula 1 is selected from thefollowing:

-   (3R)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-met    hyl-pentan-2-ol;-   (3S)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-met    hyl-pentan-2-ol;-   (2S3R)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pen    tan-2-ol;-   (2R3S)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pen    tan-2-ol; and-   any optical isomer of    3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2-ol.    Pharmaceutical Compositions

A second aspect of the invention relates to a pharmaceutical compositioncomprising a compound of formula 1 admixed with a pharmaceuticallyacceptable diluent, excipient or carrier, or a mixture thereof. Eventhough the compounds of the present invention (including theirpharmaceutically acceptable salts, esters and pharmaceuticallyacceptable solvates) can be administered alone, they will generally beadministered in admixture with a pharmaceutical carrier, excipient ordiluent, particularly for human therapy. The pharmaceutical compositionsmay be for human or animal usage in human and veterinary medicine.

Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in the“Handbook of Pharmaceutical Excipients, 2^(nd) Edition, (1994), Editedby A Wade and P J Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or in addition to, the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Salts/Esters

The compounds of the present invention can be present as salts oresters, in particular pharmaceutically acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds of the inventioninclude suitable acid addition or base salts thereof. A review ofsuitable pharmaceutical salts may be found in Berge et al, J Pharm Sci,66, 1-19 (1977). Salts are formed, for example with strong inorganicacids such as mineral acids, e.g. sulphuric acid, phosphoric acid orhydrohalic acids; with strong organic carboxylic acids, such asalkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted orsubstituted (e.g., by halogen), such as acetic acid; with saturated orunsaturated dicarboxylic acids, for example oxalic, malonic, succinic,maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylicacids, for example ascorbic, glycolic, lactic, malic, tartaric or citricacid; with aminoacids, for example aspartic or glutamic acid; withbenzoic acid; or with organic sulfonic acids, such as (C₁-C₄)-alkyl- oraryl-sulfonic acids which are unsubstituted or substituted (for example,by a halogen) such as methane- or p-toluene sulfonic acid.

Esters are formed either using organic acids or alcohols/hydroxides,depending on the functional group being esterified. Organic acidsinclude carboxylic acids, such as alkanecarboxylic acids of 1 to 12carbon atoms which are unsubstituted or substituted (e.g., by halogen),such as acetic acid; with saturated or unsaturated dicarboxylic acid,for example oxalic, malonic, succinic, maleic, fumaric, phthalic ortetraphthalic; with hydroxycarboxylic acids, for example ascorbic,glycolic, lactic, malic, tartaric or citric acid; with aminoacids, forexample aspartic or glutamic acid; with benzoic acid; or with organicsulfonic acids, such as (C₁-C₄)-alkyl- or aryl-sulfonic acids which areunsubstituted or substituted (for example, by a halogen) such asmethane- or p-toluene sulfonic acid. Suitable hydroxides includeinorganic hydroxides, such as sodium hydroxide, potassium hydroxide,calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcoholsof 1-12 carbon atoms which may be unsubstituted or substituted, e.g. bya halogen).

Enantiomers/Tautomers

In all aspects of the present invention previously discussed, theinvention includes, where appropriate all enantiomers and tautomers ofcompounds of formula 1. The man skilled in the art will recognisecompounds that possess an optical properties (one or more chiral carbonatoms) or tautomeric characteristics. The corresponding enantiomersand/or tautomers may be isolated/prepared by methods known in the art.

Stereo and Geometric Isomers

Some of the compounds of the invention may exist as stereoisomers and/orgeometric isomers—e.g. they may possess one or more asymmetric and/orgeometric centres and so may exist in two or more stereoisomeric and/orgeometric forms. The present invention contemplates the use of all theindividual stereoisomers and geometric isomers of those inhibitoragents, and mixtures thereof. The terms used in the claims encompassthese forms, provided said forms retain the appropriate functionalactivity (though not necessarily to the same degree).

The present invention also includes all suitable isotopic variations ofthe agent or a pharmaceutically acceptable salt thereof. An isotopicvariation of an agent of the present invention or a pharmaceuticallyacceptable salt thereof is defined as one in which at least one atom isreplaced by an atom having the same atomic number but an atomic massdifferent from the atomic mass usually found in nature. Examples ofisotopes that can be incorporated into the agent and pharmaceuticallyacceptable salts thereof include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively. Certainisotopic variations of the agent and pharmaceutically acceptable saltsthereof, for example, those in which a radioactive isotope such as ³H or¹⁴C is incorporated, are useful in drug and/or substrate tissuedistribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with isotopes such as deuterium,i.e., ²H, may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example, increased in vivo half-life orreduced dosage requirements and hence may be preferred in somecircumstances. Isotopic variations of the agent of the present inventionand pharmaceutically acceptable salts thereof of this invention cangenerally be prepared by conventional procedures using appropriateisotopic variations of suitable reagents.

Solvates

The present invention also includes solvate forms of the compounds ofthe present invention. The terms used in the claims encompass theseforms.

Polymorphs

The invention furthermore relates to compounds of the present inventionin their various crystalline forms, polymorphic forms and (an)hydrousforms. It is well established within the pharmaceutical industry thatchemical compounds may be isolated in any of such forms by slightlyvarying the method of purification and or isolation form the solventsused in the synthetic preparation of such compounds.

Prodrugs

The invention further includes compounds of the present invention inprodrug form. Such prodrugs are generally compounds of formula 1 whereinone or more appropriate groups have been modified such that themodification may be reversed upon administration to a human or mammaliansubject. Such reversion is usually performed by an enzyme naturallypresent in such subject, though it is possible for a second agent to beadministered together with such a prodrug in order to perform thereversion in vivo. Examples of such modifications include ester (forexample, any of those described above), wherein the reversion may becarried out be an esterase etc. Other such systems will be well known tothose skilled in the art.

Administration

The pharmaceutical compositions of the present invention may be adaptedfor oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal,intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal,intravenous, nasal, buccal or sublingual routes of administration.

For oral administration, particular use is made of compressed tablets,pills, tablets, gellules, drops, and capsules. Preferably, thesecompositions contain from 1 to 250 mg and more preferably from 10-100mg, of active ingredient per dose.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilisable solutions. Thepharmaceutical compositions of the present invention may also be in formof suppositories, pessaries, suspensions, emulsions, lotions, ointments,creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Injectable forms may contain between 10-1000 mg, preferably between10-250 mg, of active ingredient per dose.

Compositions may be formulated in unit dosage form, i.e., in the form ofdiscrete portions containing a unit dose, or a multiple or sub-unit of aunit dose.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific compound employed, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

Depending upon the need, the agent may be administered at a dose of from0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, morepreferably from 0.1 to 1 mg/kg body weight.

In an exemplary embodiment, one or more doses of 10 to 150 mg/day willbe administered to the patient for the treatment of malignancy.

Therapeutic Use

The compounds of the present invention have been found to possessanti-proliferative activity and are therefore believed to be of use inthe treatment of proliferative disorders, such as cancers, leukaemias orother disorders associated with uncontrolled cellular proliferation suchas psoriasis and restenosis.

As defined herein, an anti-proliferative effect within the scope of thepresent invention may be demonstrated by the ability to inhibit cellproliferation in an in vitro whole cell assay, for example using any ofthe cell lines A549, HeLa, HT-29, MCF7, Saos-2, CCRF-CEM, HL-60 andK-562, or by showing kinase inhibition in an appropriate assay. Theseassays, including methods for their performance, are described in moredetail in the accompanying Examples. Using such assays it may bedetermined whether a compound is anti-proliferative in the context ofthe present invention.

One preferred embodiment of the present invention therefore relates tothe use of one or more compounds of the invention in the preparation ofa medicament for treating a proliferative disorder.

As used herein the phrase “preparation of a medicament” includes the useof a compound of the invention directly as the medicament in addition toits use in a screening programme for further therapeutic agents or inany stage of the manufacture of such a medicament.

The term “proliferative disorder” is used herein in a broad sense toinclude any disorder that requires control of the cell cycle, forexample cardiovascular disorders such as restenosis and cardiomyopathy,auto-immune disorders such as glomerulonephritis and rheumatoidarthritis, dermatological disorders such as psoriasis,anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria,emphysema and alopecia. In these disorders, the compounds of the presentinvention may induce apoptosis or maintain stasis within the desiredcells as required. Preferably, the proliferative disorder is a cancer orleukaemia.

In another preferred embodiment, the proliferative disorder ispsoriasis.

The compounds of the invention may inhibit any of the steps or stages inthe cell cycle, for example, formation of the nuclear envelope, exitfrom the quiescent phase of the cell cycle (G0), G1 progression,chromosome decondensation, nuclear envelope breakdown, START, initiationof DNA replication, progression of DNA replication, termination of DNAreplication, centrosome duplication, G2 progression, activation ofmitotic or meiotic functions, chromosome condensation, centrosomeseparation, microtubule nucleation, spindle formation and function,interactions with microtubule motor proteins, chromatid separation andsegregation, inactivation of mitotic functions, formation of contractilering, and cytokinesis functions. In particular, the compounds of theinvention may influence certain gene functions such as chromatinbinding, formation of replication complexes, replication licensing,phosphorylation or other secondary modification activity, proteolyticdegradation, microtubule binding, actin binding, septin binding,microtubule organising centre nucleation activity and binding tocomponents of cell cycle signalling pathways.

A further aspect of the invention relates to a method of treating aproliferative disease, said method comprising administering to a mammala therapeutically effective amount of a compound of formula 1.

In a preferred embodiment of this aspect, the proliferative disorder iscancer or leukaemia.

In an even more preferred embodiment of this aspect, the compound isadministered in an amount sufficient to inhibit at least one CDK enzyme.

Preferably, the compound of the invention is administered in an amountsufficient to inhibit at least one of CDK1, CDK2, CDK3, CDK4, CDK6,CDK7, CDK8 and/or CDK9.

More preferably, the compound of the invention is administered in anamount sufficient to inhibit at least one of CDK2 and/or CDK4.

Even more preferably, the CDK enzyme is CDK2.

In one preferred embodiment of this aspect, the compound is administeredorally.

Another aspect of the invention relates to the use of a compound offormula 1 as an anti-mitotic agent.

Yet another aspect of the invention relates to the use of a compound offormula 1 for treating a neurodegenerative disorder.

Preferably, the neurodegenerative disorder is neuronal apoptosis.

Another aspect of the invention relates to the use of a compound offormula 1 as an antiviral agent.

Thus, another aspect of the invention relates to the use of a compoundof the invention in the preparation of a medicament for treating a viraldisorder, such as human cytomegalovirus (HCMV), herpes simplex virustype 1 (HSV-1), human immunodeficiency virus type 1 (HIV-1), andvaricella zoster virus (VZV).

In a more preferred embodiment of the invention, the compound of theinvention is administered in an amount sufficient to inhibit one or moreof the host cell CDKs involved in viral replication, i.e. CDK2, CDK7,CDK8, and CDK9 [Wang D, De la Fuente C, Deng L, Wang L, Zilberman I,Eadie C, Healey M, Stein D, Denny T, Harrison L E, Meijer L, KashanchiF. Inhibition of human immunodeficiency virus type 1 transcription bychemical cyclin-dependent kinase inhibitors. J. Virol. 2001; 75:7266-7279].

As defined herein, an anti-viral effect within the scope of the presentinvention may be demonstrated by the ability to inhibit CDK2, CDK7, CDK8or CDK9.

In a particularly preferred embodiment, the invention relates to the useof one or more compounds of the invention in the treatment of a viraldisorder which is CDK dependent or sensitive. CDK dependent disordersare associated with an above normal level of activity of one or more CDKenzymes. Such disorders preferably associated with an abnormal level ofactivity of CDK2, CDK7, CDK8 and/or CDK9. A CDK sensitive disorder is adisorder in which an aberration in the CDK level is not the primarycause, but is downstream of the primary metabolic aberration. In suchscenarios, CDK2, CDK7, CDK8 and/or CDK9 can be said to be part of thesensitive metabolic pathway and CDK inhibitors may therefore be activein treating such disorders.

Another aspect of the invention relates to the use of compounds of theinvention, or pharmaceutically acceptable salts thereof, in thepreparation of a medicament for treating diabetes.

In a particularly preferred embodiment, the diabetes is type IIdiabetes.

GSK3 is one of several protein kinases that phosphorylate glycogensynthase (GS). The stimulation of glycogen synthesis by insulin inskeletal muscle results from the dephosphorylation and activation of GS.GSK3's action on GS thus results in the latter's deactivation and thussuppression of the conversion of glucose into glycogen in muscles.

Type II diabetes (non-insulin dependent diabetes mellitus) is amulti-factorial disease. Hyperglycaemia is due to insulin resistance inthe liver, muscles, and other tissues, coupled with impaired secretionof insulin. Skeletal muscle is the main site for insulin-stimulatedglucose uptake, there it is either removed from circulation or convertedto glycogen. Muscle glycogen deposition is the main determinant inglucose homeostasis and type II diabetics have defective muscle glycogenstorage. There is evidence that an increase in GSK3 activity isimportant in type II diabetes [Chen, Y. H.; Hansen, L.; Chen, M. X.;Bjorbaek, C.; Vestergaard, H.; Hansen, T.; Cohen, P. T.; Pedersen, O.Diabetes, 1994, 43, 1234]. Furthermore, it has been demonstrated thatGSK3 is over-expressed in muscle cells of type II diabetics and that aninverse correlation exists between skeletal muscle GSK3 activity andinsulin action [Nikoulina, S. E.; Ciaraldi, T. P.; Mudaliar, S.;Mohideen, P.; Carter, L.; Henry, R. R. Diabetes, 2000, 49, 263].

GSK3 inhibition is therefore of therapeutic significance in thetreatment of diabetes, particularly type II, and diabetic neuropathy.

It is notable that GSK3 is known to phosphorylate many substrates otherthan GS, and is thus involved in the regulation of multiple biochemicalpathways. For example, GSK is highly expressed in the central andperipheral nervous systems.

Another aspect of the invention therefore relates to the use ofcompounds of the invention, or pharmaceutically acceptable saltsthereof, in the preparation of a medicament for treating a CNSdisorders, for example neurodegenerative disorders.

Preferably, the CNS disorder is Alzheimer's disease.

Tau is a GSK-3 substrate which has been implicated in the etiology ofAlzheimer's disease. In healthy nerve cells, Tau co-assembles withtubulin into microtubules. However, in Alzheimer's disease, tau formslarge tangles of filaments, which disrupt the microtubule structures inthe nerve cell, thereby impairing the transport of nutrients as well asthe transmission of neuronal messages.

Without wishing to be bound by theory, it is believed that GSK3inhibitors may be able to prevent and/or reverse the abnormalhyperphosphorylation of the microtubule-associated protein tau that isan invariant feature of Alzheimer's disease and a number of otherneurodegenerative diseases, such as progressive supranuclear palsy,corticobasal degeneration and Pick's disease. Mutations in the tau genecause inherited forms of fronto-temporal dementia, further underscoringthe relevance of tau protein dysfunction for the neurodegenerativeprocess [Goedert, M. Curr. Opin. Gen. Dev., 2001, 11, 343].

Another aspect of the invention relates to the use of compounds of theinvention, or pharmaceutically acceptable salts thereof, in thepreparation of a medicament for treating bipolar disorder.

Yet another aspect of the invention relates to the use of compounds ofthe invention, or pharmaceutically acceptable salts thereof, in thepreparation of a medicament for treating a stroke.

Reducing neuronal apoptosis is an important therapeutic goal in thecontext of head trauma, stroke, epilepsy, and motor neuron disease[Mattson, M. P. Nat. Rev. Mol. Cell. Biol., 2000, 1, 120]. Therefore,GSK3 as a pro-apoptotic factor in neuronal cells makes this proteinkinase an attractive therapeutic target for the design of inhibitorydrugs to treat these diseases.

Yet another aspect of the invention relates to the use of compounds ofthe invention, or pharmaceutically acceptable salts thereof, in thepreparation of a medicament for treating alopecia.

Hair growth is controlled by the Wnt signalling pathway, in particularWnt-3. In tissue-culture model systems of the skin, the expression ofnon-degradable mutants of β-catenin leads to a dramatic increase in thepopulation of putative stem cells, which have greater proliferativepotential [Zhu, A. J.; Watt, F. M. Development, 1999, 126, 2285]. Thispopulation of stem cells expresses a higher level ofnon-cadherin-associated β-catenin [DasGupta, R.; Fuchs, E. Development,1999, 126, 4557], which may contribute to their high proliferativepotential. Moreover, transgenic mice overexpressing a truncatedβ-catenin in the skin undergo de novo hair-follicle morphogenesis, whichnormally is only established during embryogenesis. The ectopicapplication of GSK3 inhibitors may therefore be therapeutically usefulin the treatment of baldness and in restoring hair growth followingchemotherapy-induced alopecia. A further aspect of the invention relatesto a method of treating a GSK3-dependent disorder, said methodcomprising administering to a subject in need thereof, a compoundaccording to the invention, or a pharmaceutically acceptable saltthereof, as defined above in an amount sufficient to inhibit GSK3.

Preferably, the compound of the invention, or pharmaceuticallyacceptable salt thereof, is administered in an amount sufficient toinhibit GSK3p.

In one embodiment of the invention, the compound of the invention isadministered in an amount sufficient to inhibit at least one PLK enzyme.

The polo-like kinases (PLKs) constitute a family of serine/threonineprotein kinases. Mitotic Drosophila melanogaster mutants at the pololocus display spindle abnormalities [Sunkel et al., J. Cell Sci., 1988,89, 25] and polo was found to encode a mitotic kinase [Llamazares etal., Genes Dev., 1991, 5, 2153]. In humans, there exist three closelyrelated PLKs [Glover et al., Genes Dev., 1998, 12, 3777]. They contain ahighly homologous amino-terminal catalytic kinase domain and theircarboxyl termini contain two or three conserved regions, the polo boxes.The function of the polo boxes remains incompletely understood but theyare implicated in the targeting of PLKs to subcellular compartments [Leeet al., Proc. Natl. Acad. Sci. USA, 1998, 95, 9301; Leung et al., Nat.Struct. Biol., 2002, 9, 719], mediation of interactions with otherproteins [Kauselmann et al., EMBO J., 1999, 18, 5528], or may constitutepart of an autoregulatory domain [Nigg, Curr. Opin. Cell Biol., 1998,10, 776]. Furthermore, the polo box-dependent PLK1 activity is requiredfor proper metaphase/anaphase transition and cytokinesis [Yuan et al.,Cancer Res., 2002, 62, 4186; Seong et al., J. Biol. Chem., 2002, 277,32282].

Studies have shown that human PLKs regulate some fundamental aspects ofmitosis [Lane et al., J. Cell. Biol., 1996, 135, 1701; Cogswell et al.,Cell Growth Differ., 2000, 11, 615]. In particular, PLK1 activity isbelieved to be necessary for the functional maturation of centrosomes inlate G2/early prophase and subsequent establishment of a bipolarspindle. Depletion of cellular PLK1 through the small interfering RNA(siRNA) technique has also confirmed that this protein is required formultiple mitotic processes and completion of cytokinesis [Liu et al.,Proc. Natl. Acad. Sci. USA, 2002, 99, 8672].

In a more preferred embodiment of the invention, the compound of theinvention is administered in an amount sufficient to inhibit PLK1.

Of the three human PLKs, PLK1 is the best characterized; it regulates anumber of cell division cycle effects, including the onset of mitosis[Toyoshima-Morimoto et al., Nature, 2001, 410, 215; Roshak et al., Cell.Signalling, 2000, 12, 405], DNA-damage checkpoint activation [Smits etal., Nat. Cell Biol., 2000, 2, 672; van Vugt et al., J. Biol. Chem.,2001, 276, 41656], regulation of the anaphase promoting complex [Sumaraet al., Mol. Cell, 2002, 9, 515; Golan et al., J. Biol. Chem., 2002,277, 15552; Kotani et al., Mol. Cell, 1998, 1, 371], phosphorylation ofthe proteasome [Feng et al., Cell Growth Difer., 2001, 12, 29], andcentrosome duplication and maturation [Dai et al., Oncogene, 2002, 21,6195].

Specifically, initiation of mitosis requires activation of M-phasepromoting factor (MPF), the complex between the cyclin dependent kinaseCDK1 and B-type cyclins [Nurse, Nature, 1990, 344, 503]. The latteraccumulate during the S and G2 phases of the cell cycle and promote theinhibitory phosphorylation of the MPF complex by WEE1, MIK1, and MYT1kinases. At the end of the G2 phase, corresponding dephosphorylation bythe dual-specificity phosphatase CDC25C triggers the activation of MPF[Nigg, Nat. Rev. Mol. Cell. Biol., 2001, 2, 21]. In interphase, cyclin Blocalizes to the cytoplasm [Hagting et al., EMBO J., 1998, 17, 4127], itthen becomes phosphorylated during prophase and this event causesnuclear translocation [Hagting et al., Curr. Biol., 1999, 9, 680; Yanget al., J. Biol. Chem., 2001, 276, 3604]. The nuclear accumulation ofactive MPF during prophase is thought to be important for initiatingM-phase events [Takizawa et al., Curr. Opin. Cell Biol., 2000, 12, 658].However, nuclear MPF is kept inactive by WEE1 unless counteracted byCDC25C. The phosphatase CDC25C itself, localized to the cytoplasm duringinterphase, accumulates in the nucleus in prophase [Seki et al., Mol.Biol. Cell, 1992, 3, 1373; Heald et al., Cell, 1993, 74, 463; Dalal etal., Mol. Cell. Biol., 1999, 19, 4465]. The nuclear entry of both cyclinB [Toyoshima-Morimoto et al., Nature, 2001, 410, 215] and CDC25C[Toyoshima-Morimoto et al., EMBO Rep., 2002, 3, 341] are promotedthrough phosphorylation by PLK1 [Roshak et al., Cell. Signalling, 2000,12, 405]. This kinase is an important regulator of M-phase initiation.

In one particularly preferred embodiment, the compounds of the inventionare ATP-antagonistic inhibitors of PLK1.

In the present context ATP antagonism refers to the ability of aninhibitor compound to diminish or prevent PLK catalytic activity, i.e.phosphotransfer from ATP to a macromolecular PLK substrate, by virtue ofreversibly or irreversibly binding at the enzyme's active site in such amanner as to impair or abolish ATP binding.

In another preferred embodiment, the compound of the invention isadministered in an amount sufficient to inhibit PLK2 and/or PLK3.

Mammalian PLK2 (also known as SNK) and PLK3 (also known as PRK and FNK)were originally shown to be immediate early gene products. PLK3 kinaseactivity appears to peak during late S and G2 phase. It is alsoactivated during DNA damage checkpoint activation and severe oxidativestress. PLK3 also plays an important role in the regulation ofmicrotubule dynamics and centrosome function in the cell and deregulatedPLK3 expression results in cell cycle arrest and apoptosis [Wang et al.,Mol. Cell. Biol., 2002, 22, 3450]. PLK2 is the least well understoodhomologue of the three PLKs. Both PLK2 and PLK3 may have additionalimportant post-mitotic functions [Kauselmann et al., EMBO J., 1999, 18,5528].

Another aspect of the invention relates to the use of a compound offormula 1 for inhibiting a protein kinase.

In a preferred embodiment of this aspect, the protein kinase is a cyclindependent kinase. Preferably, the protein kinase is CDK1, CDK2, CDK3,CDK4, CDK6, CDK7, CDK8 or CDK9, more preferably CDK2.

A further aspect of the invention relates to a method of inhibiting aprotein kinase, said method comprising contacting said protein kinasewith a compound of formula 1.

In a preferred embodiment of this aspect, the protein kinase is a cyclindependent kinase, even more preferably CDK2.

Assays

Another aspect of the invention relates to the use of a compound asdefined hereinabove in an assay for identifying further candidatecompounds that influence the activity of one or more CDK enzymes.

Preferably, the assay is capable of identifying candidate compounds thatare capable of inhibiting one or more CDK enzymes.

More preferably, the assay is a competitive binding assay.

Preferably, the candidate compound is generated by conventional SARmodification of a compound of the invention.

As used herein, the term “conventional SAR modification” refers tostandard methods known in the art for varying a given compound by way ofchemical derivatisation. Thus, in one aspect, the identified compoundmay act as a model (for example, a template) for the development ofother compounds. The compounds employed in such a test may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The abolition of activity or the formation ofbinding complexes between the compound and the agent being tested may bemeasured.

The assay of the present invention may be a screen, whereby a number ofagents are tested. In one aspect, the assay method of the presentinvention is a high through-put screen.

This invention also contemplates the use of competitive drug screeningassays in which neutralising antibodies capable of binding a compoundspecifically compete with a test compound for binding to a compound.

Another technique for screening provides for high throughput screening(HTS) of agents having suitable binding affinity to the substances andis based upon the method described in detail in WO 84/03564.

It is expected that the assay methods of the present invention will besuitable for both small and large-scale screening of test compounds aswell as in quantitative assays.

Preferably, the competitive binding assay comprises contacting acompound of formula 1 with a CDK enzyme in the presence of a knownsubstrate of said CDK enzyme and detecting any change in the interactionbetween said CDK enzyme and said known substrate.

A sixth aspect of the invention provides a method of detecting thebinding of a ligand to a CDK enzyme, said method comprising the stepsof:

(i) contacting a ligand with a CDK enzyme in the presence of a knownsubstrate of said CDK enzyme;

(ii) detecting any change in the interaction between said CDK enzyme andsaid known substrate;

and wherein said ligand is a compound of formula 1.

One aspect of the invention relates to a process comprising the stepsof:

(a) performing an assay method described hereinabove;

(b) identifying one or more ligands capable of binding to a ligandbinding domain; and

(c) preparing a quantity of said one or more ligands.

Another aspect of the invention provides a process comprising the stepsof:

(a) performing an assay method described hereinabove;

(b) identifying one or more ligands capable of binding to a ligandbinding domain; and

(c) preparing a pharmaceutical composition comprising said one or moreligands.

Another aspect of the invention provides a process comprising the stepsof:

(a) performing an assay method described hereinabove;

(b) identifying one or more ligands capable of binding to a ligandbinding domain;

(c) modifying said one or more ligands capable of binding to a ligandbinding domain;

(d) performing the assay method described hereinabove;

(e) optionally preparing a pharmaceutical composition comprising saidone or more ligands.

The invention also relates to a ligand identified by the methoddescribed hereinabove.

Yet another aspect of the invention relates to a pharmaceuticalcomposition comprising a ligand identified by the method describedhereinabove.

Another aspect of the invention relates to the use of a ligandidentified by the method described hereinabove in the preparation of apharmaceutical composition for use in the treatment of proliferativedisorders.

The above methods may be used to screen for a ligand useful as aninhibitor of one or more CDK enzymes.

Process

A further aspect of the invention relates to a process for preparing acompound of formula I as defined hereinabove, said process comprisingreacting a compound of formula V with a compound of formula VI

wherein R¹⁻⁹ are as defined in claim 1 and X is Cl or F.

Preferably, said compound of formula V is prepared by the followingsteps:

-   (i) reacting a compound of formula II with a compound of formula III    to form a compound of formula IV;-   (ii) alkylating said compound of formula IV with an alkyl halide,    R⁵—X′, to form a compound of formula V.

Preferably, said compound of formula VI is prepared by the followingsteps:

-   (i) oxidising a compound of formula VIII, wherein PG is a protecting    group, to form a compound of formula IX;-   (ii) alkylating said compound of formula IX to form a compound of    formula X;-   (iii) removing protecting group PG from said compound of formula X    to form a compound of formula IX, which is equivalent to formula VI    (where one of R³ or R⁴ is H).

Alternatively, said compound of formula VI is prepared by the followingsteps:

-   (i) oxidising a compound of formula VIII, wherein PG is a protecting    group, to form a compound of formula IX;-   (ii) alkylating said compound of formula IX to form a compound of    formula X;-   (iii) oxidising said compound of formula X to form a compound of    formula XI;-   (iv) alkylating said compound of formula XI to form a compound of    formula XII;-   (v) removing protecting group PG from said compound of formula XIII    to form a compound of formula VI.

More preferably, the oxidation in steps (i) and (iii) of the aboveprocesses are achieved by means of a Swern oxidation.

Preferably, the alkylation reaction of steps (ii) and (iv) of the aboveprocesses are achieved by treating the compound with an alkyllithiumreagent in the presence of a copper bromide/dimethyl sulfide complexcatalyst.

In alternative preferred embodiment, R³═R⁴ and said compound of formulaVI is prepared by a process which comprises the steps of:

-   (i) converting a compound of formula XVI, where PG′ is a protecting    group and R is an alkyl group, to a compound of formula XVII;-   (ii) removing protecting groups PG′ from said compound of formula    XVII to form a compound of formula VI.

Preferably, said compound of formula XVI is converted to a compound offormula XVII via a double Grignard reaction.

More preferably, in respect of this embodiment, said compound of formulaVI is prepared by the following steps:

-   (i) reacting a compound of formula XIV with SOCl₂ and MeOH to form a    compound of formula XV;-   (ii) protecting the amino group of said compound of formula XV to    form a compound of formula XVIa;-   (iii) reacting said compound of formula XVIa with a Grignard reagent    R³X″, where R³ is as defined in claim 1 and X″ is a halide, to form    a compound of formula XVII;-   (iv) removing protecting groups PG′ from said compound of formula    XVII to form said compound of formula VI.

Suitable protecting groups PG and PG′ will be familiar to those skilledin the relevant art. By way of example, preferably protecting group PG′is a benzyl group, Bn, and protecting group PG is a trityl group.

Further details of the preparation of compounds the present inventionare outlined in the accompanying Examples under the heading “Synthesis”.

The present invention is further described by way of the followingexamples.

EXAMPLES

In contrast to roscovitine, the compounds of the present inventioncontain modified purine C-2 substituents. In particular, the compoundsof the invention contain C-2 substituents having a secondary or tertiaryalcohol group rather than a primary alcohol group. Without wishing to bebound by theory, it is believed that the presence of such modified C-2substituents leads to a reduction in the metabolic alcohol-carboxylconversion.

In order to offset the reduction in aqueous solubility expected as aresult of incorporating additional alkyl substituents into the C-2substituent, the C-6 benzylamino group of roscovitine was replaced witha (pyridin-3-yl)-methylamino group. The accompanying examplesdemonstrate that this modification is tolerated in terms of biologicalactivity (CDK2/cyclin E or A, CDK1/cyclin B inhibition andanti-proliferative effect on human tumour cell line).

Thus, the present invention demonstrates that modification of the purineC-2 and C-6 substituents of roscovitine affords novel compounds withenhanced therapeutic utility. Indeed, it has been shown that placementof one or two lower alkyl substituents at the carbinol C of the purineC-2 substituent present in roscovitine is not only tolerated in terms ofretaining the desired biological activity (potency and selectivity ofprotein kinase inhibition; cytotoxicity), but in some cases providesmore potent compounds. Moreover, the inclusion of a(pyridin-3-yl)-methylamino group in place of the benzylamino groupensures improved hydrophilicity and aqueous solubility profiles for thecompounds of this invention compared to roscovitine (calculatedn-octanol/water partition coefficients: 2.5<ClogP<3.8 compared toClogP=3.7 for roscovitine). Furthermore, selected compounds exemplifiedherein have been shown to possess enhanced resistance to metabolicdegradation using an appropriate in vitro model system.

Synthesis

The compounds of general structure 1 can be prepared by methods known inthe art (reviewed in Fischer, P. M., and Lane, D. P. Curr. Med. Chem.2001, 7, 1213-1245). A convenient synthetic route is shown below inScheme 1 and starts with commercially available 2,6-dichloropurine (2,X═Cl) or 2-amino-6-chloropurine (2, X═NH₂). In the latter case, theamino group is transformed to provide the particularly suitable6-chloro-2-fluoro-purine starting material (2, X═F; Gray, N. S., Kwon,S., and Schultz, P. G. Tetrahedron Lett. 1997, 38, 1161-1164.).Selective amination at the more reactive C-6 position with theappropriate pyridylmethylamine 3 then affords intermediate 4. This isalkylated at the N-9 position, e.g. by nucleophilic substitution usingthe appropriate alkyl halide R⁵—X. The product 5 is finally aminatedwith a hydroxyethylamine 6 at elevated temperature.

Substituted amino alcohols 6 (R¹ or R²

H) can be synthesized from α-amino alcohols 7 (R¹ or R²

H) as shown below in Scheme 2. Many of the latter are availablecommercially; alternatively, they can be prepared readily by reductionof the corresponding α-amino acids. The initial reaction in thesynthetic methodology adopted was trityl protection of the aminofunction to afford intermediate 8 (R¹ or R²

H; Evans, P. A., Holmes, A. B., and Russell, K. J. Chem. Soc., PerkinTrans. 1, 1994, 3397-3409). This was submitted to Swern oxidation to thecorresponding aldehyde 9 (R¹ or R²

H; Takayama, H., Ichikawa, T., Kuwajima, T., Kitajima, M., Seki, H.,Aimi, N., and Nonato, M. G. J. Am. Chem. Soc. 2000, 122, 8635-8639).Introduction of the substituent R³ (if R²

H) or R⁴ (if R¹

H) was accomplished via chelation-controlled alkylation (Reetz, M. T.,Roelfing, K., and Griebenow, N. Tetrahedron Lett. 1994, 35, 1969-1972)using the appropriate alkyllithium reagent and a copper bromide/dimethylsulfide complex catalyst in diethyl ether. Depending on the substituentto be introduced, this procedure afforded intermediates 10 indiastereomeric excess (de) of 50-80%. Alternatively, achiral methods canbe used, optionally followed by separation/resolution of the opticalisomers. For production of amino alcohols where both R³ and R⁴ are otherthan H, intermediate 10 was subjected to another Swern oxidationreaction to the respective ketone 12, followed by introduction of thesecond substituent through alkylation. The final step in the synthesisfor all the amino alcohols was removal of the trityl group usingtrifluoroacetic acid to afford 6 or 11.

In those cases where amino alcohols contain two identical substituentsat the carbinol C (6, R¹ or R²<>H; R³═R⁴, not H), these can be obtaineddirectly from a suitable corresponding α-amino acid ester, e.g. bydouble Grignard alkylation (Guenther, B. R., and Kirmse, W. Liebigs Ann.Chem. 1980, 518-532).

Kinase Assays

The compounds from the examples below were investigated for theirCDK2/cyclin E, CDK1/cyclin B, CDK4/cyclin D1 and CDK7/cyclin H, ERK-2,and PKA inhibitory activity. His₆-tagged recombinant humancyclin-dependent kinases CDK1/cyclin B1, CDK2/cyclin E, CDK4 andCDK7/cyclin H were expressed in sf9 cells using a baculovirus expressionsystem. Recombinant cyclin D1 was expressed in E. coli.

Proteins were purified by metal chelate affinity chromatography togreater than 90% homogeneity. Kinase assays were performed in 96-wellplates using recombinant CDK/cyclins, recombinant active ERK-2 (UpstateBiotechnology), or cyclic AMP-dependent kinase (PKA) catalytic subunit(Calbiochem Cat. 539487). Assays were performed in assay buffer (25 mMβ-glycerophosphate, 20 mM MOPS, 5 mM EGTA, 1 mM DTT, 1 mM Na₃VO₃, pH7.4), into which were added 2-4 μg of active enzyme with appropriatesubstrates (purified histone H1 for CDK2, recombinant GST-retinoblastomaprotein (residues 773-928) for CDK4,biotinyl-Ahx-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)₄ peptide for CDK7, myelinbasic protein for ERK-2, or peptide Kemptide (Fluka Biochemika Cat.60645) for PKA). The reaction was initiated by addition of Mg/ATP mix(15 mM MgCl₂+100 μM ATP with 30-50 kBq per well of [γ-³²P]-ATP) andmixtures incubated for 10 min (CDK2/cyclin E, ERK-2, PKA) or 45 min(CDK4/cyclin D1, CDK7/cyclin H) as required, at 30° C. Reactions werestopped on ice, followed by filtration through p81 filterplates or GF/Cfilterplates (for CDK4) (Whatman Polyfiltronics, Kent, UK), except forCDK7 where, after stopping reaction on ice, 10 μL of 10 mg/mL avidin wasadded to each well and further incubated for 10 min followed byfiltration as per CDK2 assay. After washing 3 times with 75 mM aqorthophosphoric acid, plates were dried, scintillant added andincorporated radioactivity measured in a scintillation counter(TopCount, Packard Instruments, Pangbourne, Berks, UK). Compounds forkinase assay were made up as 10 mM stocks in DMSO and diluted into 10%DMSO in assay buffer. Data was analysed using curve-fitting software(GraphPad Prism version 3.00 for Windows, GraphPad Software, San DiegoCalif. USA) to determine IC₅₀ values (concentration of test compoundwhich inhibits kinase activity by 50%.). These values for the compoundsof the present invention are shown in Table 1.

MTT Cytotoxicity Assay

The compounds from the examples below were subjected to a standardcellular proliferation assay using the following human tumour celllines: A549, HeLa, HT-29, MCF₇, Saos-2, CCRF-CEM, HL-60, and K-562. Thecell lines were obtained from the ATCC (American Type CultureCollection, 10801 University Boulevard, Manessas, Va. 20110-2209, USA).Standard 72-h MTT (thiazolyl blue;3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assayswere performed (Haselsberger, K.; Peterson, D. C.; Thomas, D. G.;Darling, J. L. Anti Cancer Drugs 1996, 7, 331-8; Loveland, B. E.; Johns,T. G.; Mackay, I. R.; Vaillant, F.; Wang, Z. X.; Hertzog, P. J.Biochemistry International 1992, 27, 501-10). In short: cells wereseeded into 96-well plates according to doubling time and incubatedovernight at 37° C. Test compounds were made up in DMSO and a ⅓ dilutionseries prepared in 100 μL cell media, added to cells (in triplicates)and incubated for 72 ho at 37° C. MTT was made up as a stock of 5 mg/mLin cell media and filter-sterilised. Media was removed from cellsfollowed by a wash with 200 μL PBS. MTT solution was then added at 20 μLper well and incubated in the dark at 37° C. for 4 h. MTT solution wasremoved and cells again washed with 200 μL PBS. MTT dye was solubilisedwith 200 μL per well of DMSO with agitation. Absorbance was read at 540nm and data analysed using curve-fitting software (GraphPad Prismversion 3.00 for Windows, GraphPad Software, San Diego Calif. USA) todetermine IC₅₀ values (concentration of test compound which inhibitscell growth by 50%). These values for the compounds of the presentinvention are shown in Table 2.

Selective Cytotoxicity

Representative compounds of this invention were examined for theiranti-proliferative effect against human tumour cell lines andimmortalized non-transformed human cell lines (“normal” cell lines). Theresults are summarized in Table 3. It can be seen that the compoundsexamined are several-fold more potent anti-proliferative agents comparedto roscovitine. Furthermore, their cytotoxic effect is significantlymore pronounced against transformed versus non-transformed cell lines.

Comparative In Vitro Metabolism Assay

Microsomal Incubations and Preparation of Samples for Analysis

Microsomes were obtained from Totem Biologicals, Northampton, England.Microsomal protein (0.2 mg) and roscovitine or a test compound of thisinvention (final concentration 10 μM) were mixed in phosphate-bufferedsaline (100 μL) containing NADPH, (20 mM), MgCl₂ (10 mM), and EDTA (1.5mM). Samples were incubated for 30 min and the reaction stopped by theaddition of ice-cold methanol (300 μL) containing olomoucine (Vesely,J., Havlicek, L., Strnad, M., Blow, J. J., Donella-Deana, A., Pinna, L.,Letham, D. S., Kato, J., Detivaud, L., Leclerc, S., Meijer, L. Eur. J.Biochem. 1994, 224, 771-786) as internal standard. Calibration curveswere prepared at 0, 1, and 10 μM in microsomes pre-incubated for 30 minand these were also treated with methanol containing olomoucine. Allsamples were then centrifuged and the supernatants analysed by liquidchromatography-mass spectrometry.

Liquid Chromatography-Mass Spectrometry

The chromatography column was a Supelco LC-ABZ, 50×4.6 mm, 5 μmzwitterionic column (Supelco Inc., Supelco Park, Bellefonte, Pa., USA).Gradient eluants consisted of methanol (A) and 0.1% formic acid in water(B). The gradient started with 10:90 (A:B v/v) which was heldisocratically for 0.5 min, followed by a linear increase to 90:10 (A:Bv/v) over 6 min which was then held at these conditions for a further 4min. The flow rate was 1 mL/min throughout. For LC-UV-MS samples wereintroduced using a Gilson 215 autosampler (Anachem Ltd., Bedfordshire,UK) attached to a Thermoseparations P4000 quaternary pump, column (asdescribed above) and Thermoseparations UV 1000 detector set to 254 nm(Thermoquest Ltd., Hemel Hempstead, Hertfordshire, UK). Eluant from thedetector passed, without splitting, into a Thermoquest LCQ ion trap massspectrometer fitted with an electrospray source operated in positivemode. Mass spectrometer conditions were sheath gas 80, auxiliary gas 20(both arbitrary units), capillary voltage 4 to 4.5 kV and heatedcapillary temperature 250 to 280° C. The mass range was 50-750. Scantime was controlled by the ion trap which was set to a maximum ioninjection time of 200 ms or the time required to inject 2×10⁸ ions; foreach scan the system automatically used whichever time was reachedfirst.

Data Analysis

To analyse the results selected ion traces of the MH⁺ ions of the testcompound and internal standard were extracted and the area of therelevant peaks obtained. The peak area ration (test compound/internalstandard) of the test incubation was then compared with the peak arearatios obtained fro the calibration curve of the test compound. Fromthese values the concentration of test compound remaining after 30 minmicrosomal protein incubation was determined. Results for representativecompounds of the present invention are summarized in Table 3, wherecompound metabolic stability is also compared with that of roscovitinein terms of metabolism (column A), in vitro CDK2 inhibition (column B),and in vitro cytotoxicity on tumour cell lines (column C). Comparativein vitro efficacy (column A×C) and cellular exposure (column A×C) arealso shown. These results suggest that the compounds of the presentinvention will have improved in vivo efficacy compared to roscovitine.Calculated n-octanol/water partition coefficients (ClogP) are alsoincluded in Table 3. It can be seen that those compounds with improvedcellular activity and metabolic stability also possess lower ClogP thanroscovitine, suggesting improved aqueous solubility and thus ease offormulation for drug administration in vivo.

(2R)-2-(6-Benzylamino-9-isopropyl-9H-purin-2-ylamino)-butyric acid

Benzyl-(2-fluoro-9-isopropyl-9H-purin-6-yl)-amine (151 mg, 0.5 mmol) wasdissolved in NMP (5 mL) and DBU (1.5 mL, 10 mmol).(R)-(−)-2-Aminobutyric acid (99% ee/GLC; 1.03 g, 10 mmol) was then addedand the mixture was stirred under N₂ at 160° C. for 1 h. After cooling,the mixture was diluted with citric acid (10% aq solution) and CH₂Cl₂(25 mL each). The phases were separated and the organic fraction wasextracted with brine (2×10 mL), dried over MgSO₄, filtered, andevaporated. The residue was redissolved in MeCN and was fractionated bypreparative RP-HPLC (Vydac 218TP1022, 9 mL/min, 22.5-32.5% MeCN in H₂Ocontaining 0.1% CF₃COOH over 40 min). Appropriate fractions were pooledand lyophilised to afford the pure title compound (137 mg, 74.4%) as anamorphous off-white solid. Anal. RP-HPLC (Vydac 218TP54, 1 mL/min):t_(R)=16.04 min (0-60% MeCN), 15.95 min (22.5-32.5% MeCN in H₂Ocontaining 0.1% CF₃COOH over 20 min), purity: >98% (λ=214 nm). ¹H-NMR(d₆-DMSO, 300 MHz) δ: 0.95 (t, J=7.3 Hz, 3H, CH₂CH ₃); 1.51 (d, J=6.7Hz, 6H, CH(CH ₃)₂); 1.78 (m, J=7.3 Hz, 2H, CH ₂CH₃); 4.27 (m, 1H,CHCH2); 4.64 (hept., J=6.7 Hz, 1H, CH(CH ₃)₂); 4.69 (m, 2H, CH ₂Ph);7.25-7.41 (m, 6H, ArH). DE-MALDI-TOF MS (α-cyano-4-hydroxycinnamic acidmatrix): [M+H]⁺=369.41. FAB-MS: [M+H]⁺=369.2033 (C₁₉H₂₅N₆O₂ requires369.2039).

(R)-2-(Trityl-amino)-butan-1-ol

To a stirred solution of (R)-(−)-2-aminobutan-1-ol (10 g, 1 eq, 112.18mmol) in DCM (500 mL) under an argon atmosphere at room temperature, wasadded DIEA (30 mL, 1.54 eq, 172.22 mmol) followed by trityl chloride(35.4 mL, 1.13 eq, 126.98 mmol). The reaction mixture was stirred atroom temperature for 48 h, when TLC (hexane:ether:MeOH; 55:40:5)indicated that the reaction had gone to completion. The solvent wasevaporated in vacuo and the residue precipitated from acetone (50 mL)with hexane (900 mL) with stirring, the precipitate was removed byfiltration and the filtrate was evaporated in vacuo. The residue wasdissolved in hexane (1 L), filtered, and the filtrate was evaporated invacuo to afford the title compound as a light yellow oil. Yield: 32 g(86%). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.56 (t, 3H, J=7.41 Hz, —NHCH(CH₂ CH₃)CH₂OH), 1.10 (m, 2H, —NHCH(CH₂ CH₃ )CH₂OH), 2.22 (m, 1H,—NHCH(CH₂CH₃)CH₂ OH), 2.38 (m, 1H, —NHCH(CH₂CH₃)CH₂OH), 2.72+3.00 (2×m,2H, —NHCH(CH₂CH₃)CH₂ OH), 4.28 (t, 1H, J=5.26 Hz, —NHCH(CH₂CH₃) CH₂OH),7.14-7.49 (m, 15H, 3×Ph).

(S)-2-(Trityl-amino)-butan-1-ol

To a stirred solution of (S)-(+)-2-aminobutan-1-ol (10 g, 1 eq, 112.18mmol) in DCM (500 mL) under an argon atmosphere at room temperature, wasadded DIEA (30 mL, 1.54 eq, 172.22 mmol) followed by trityl chloride(35.4 mL, 1.13 eq, 126.98 mmol). The reaction mixture was stirred atthis temperature for 48 h, when TLC (hexane:ether:MeOH; 55:40:5)indicated that the reaction had gone to completion. The solvent wasevaporated in vacuo and the residue precipitated from acetone (50 mL)with hexane (900 mL) with stirring, the precipitate was removed byfiltration and the filtrate was evaporated in vacuo. The residue wasdissolved in hexane (1 L), filtered, and the filtrate was evaporated invacuo to afford the title compound as a light yellow oil. Yield: 33 g(89%). ¹H-NMR (d₆-DMSO, 250 MHz): δ 0.58 (t, 3H, J=7.26 Hz, —NHCH(CH₂CH₃ )CH₂OH), 1.10 (m, 2H, —NHCH(CH₂ CH₃)CH₂OH), 2.24 (m, 1H,—NHCH(CH₂CH₃)CH₂ OH), 2.39 (m, 1H, —NHCH(CH₂CH₃)CH₂OH), 2.76 & 3.03(2×m, 2H, —NHCH(CH₂CH₃)CH₂ OH), 4.32 (t, 1H, J=4.97 Hz, —NHCH(CH₂CH₃)CH₂OH), 7.15-7.52 (m, 15H, 3×Ph).

(R)-2-(Trityl-amino)-butyraldehyde

To a stirred solution of DMSO (3.0 mL, 2.8 eq, 42.28 mmol) in DCM (30mL) under an argon atmosphere at −45° C., was added oxalyl chloride (2 Min DCM, 10.56 mL, 1.40 eq, 21.12 mmol), dropwise. The reaction mixturewas stirred at −45° C. for 1 h, after which time a solution of(R)-2-(trityl-amino)-butan-1-ol (5 g, 1 eq, 15.08 mmol) in DCM (30 mL)was added dropwise with stirring. The reaction mixture was stirred atthis temperature for 3 h, when TLC (hexane:ether; 80:20) indicated thatthe reaction had gone to completion. To the reaction mixture was added asolution of TEA (10.5 mL, 5 eq, 75.33 mmol) in DCM (30 mL), and thesolution allowed to warm to room temperature over 16 h. The reactionmixture was diluted with more DCM (200 mL) and washed with water (250mL). The aqueous phase was extracted with DCM (3×50 mL), and thecombined organic phase washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was dissolved in ether (30 mL), thesolid precipitate removed by filtration and the filtrate was evaporatedin vacuo. The residue was dissolved in hexane (50 mL), the solidprecipitate removed by filtration and the filtrate was evaporated invacuo to afford the title compound as a light yellow oil. Yield: 2.59 g(52%). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.77 (t, 3H, J=7.42 Hz, —NHCH(CH₂ CH₃)CHO), 1.34-1.61 (m, 2H, —NHCH(CH₂ CH₃)CHO), 2.92 (m, 1H,—NHCH(CH₂CH₃)CHO), 3.62 (d, 1H, J=8.21 Hz, —NHCH(CH₂CH₃)CHO), 7.16-7.46(m, 15H, 3×Ph), 8.77 (d, 1H, J=3.00 Hz, —NHCH(CH₂CH₃)CHO).

(S)-2-(Trityl-amino)-butyraldehyde

To a stirred solution of DMSO (2.4 mL, 2.8 eq, 33.82 mmol) in DCM (30mL) under an argon atmosphere at −45° C., was added oxalyl chloride (2 Min DCM, 8.45 mL, 1.40 eq, 16.9 mmol), dropwise. The reaction mixture wasstirred at −45° C. for 1 h, after which time a solution of(S)-2-(trityl-amino)-butan-1-ol (4 g, 1 eq, 12.07 mmol) in DCM (30 mL)was added dropwise with stirring. The reaction mixture was stirred atthis temperature for 3 h, when TLC (hexane:ether; 80:20) indicated thatthe reaction had gone to completion. To the reaction mixture was added asolution of TEA (8.4 mL, 5 eq, 60.27 mmol) in DCM (30 mL), and thesolution allowed to warm to room temperature over 16 h. The reactionmixture was diluted with more DCM (100 mL) and washed with water (250mL). The aqueous phase was extracted with DCM (3×50 mL), and thecombined organic phase washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was dissolved in ether (30 mL), thesolid precipitate removed by filtration and the filtrate was evaporatedin vacuo. The residue was dissolved in hexane (50 mL), the solidprecipitate removed by filtration and the filtrate was evaporated invacuo to afford the title compound as a light yellow oil. Yield: 3.64 g(91%). ¹H-NMR (d₆-DMSO, 250 MHz): δ 0.77 (t, 3H, J=7.42 Hz, —NHCH(CH₂CH₃ )CHO), 1.37-1.59 (m, 2H, —NHCH(CH₂ CH₃)CHO), 2.93 (m, 1H,—NHCH(CH₂CH₃)CHO), 3.62 (d, 1H, J=5.84 Hz, —NHCH(CH₂CH₃)CHO), 7.16-7.46(m, 15H, 3×Ph), 8.77 (d, 1H, J=3.00 Hz, —NHCH(CH₂CH₃)CHO).

(2S,3R)-3-(Trityl-amino)-pentan-2-ol

To a stirred suspension of CuBr.SMe₂ (2.74 g, 2.2 eq, 13.33 mmol) inEt₂O (100 mL) under an argon atmosphere at −70° C., was addedmethyllithium (1.6 M in Et₂O, 16.6 mL, 4.4 eq, 26.56 mmol) dropwise, andthe solution allowed to warm to room temperature. The mixture wasrecooled to −70° C., to which was added a solution of(R)-2-(trityl-amino)-butyraldehyde (2 g, 1 eq, 6.05 mmol) in Et₂O (25mL) dropwise with stirring. The reaction mixture was stirred at thistemperature for 2 h, when TLC (hexane:ether; 80:20) indicated that thereaction had gone to completion. To the reaction mixture was added asaturated aqueous solution of NH₄Cl (100 mL) and allowed to warm to roomtemperature over 16 h. The reaction mixture was extracted with ether(2×200 mL), and the combined organic phase washed with brine (50 mL),dried (MgSO₄) and evaporated in vacuo. The residue was purified bysilica gel column chromatography, eluted with hexane:ether (80:20) toafford the title compound as a light yellow oil. Yield: 1.91 g (91%).(80% de 2S,3R: 20% de 2R,3R). ¹H-NMR (d₆-DMSO, 250 MHz): δ 0.47 & 0.55(2×t, J=7.43 & 7.27 Hz, —NHCH(CH₂ CH₃ )CH(CH₃)OH), 0.99-1.12 (m, 5H,—NHCH(CH₂CH₃)CH(CH₃ )OH), 2.03 (m, 1H, —NHCH(CH₂CH₃)CH(CH₃)OH),3.32-3.51 (m, 1H, —NHCH(CH₂CH₃)CH(CH₃)OH), 4.40 (d, 1H, J=3.79 Hz,—NHCH(CH₂CH₃)CH(CH₃)OH), 7.14-7.51 (m, 15H, 3×Ph).

(2R,3S)-3-(Trityl-amino)-pentan-2-ol

To a stirred suspension of CuBr.SMe₂ (2.74 g, 2.2 eq, 13.33 mmol) inether (100 mL) under an argon atmosphere at −70° C., was added methyllithium (1.6 M in ether, 15.13 mL, 4.0 eq, 24.21 mmol) dropwise and thesolution allowed to warm to room temperature. The mixture was recooledto −70° C., to which was added a solution of(S)-2-(trityl-amino)-butyraldehyde (2 g, 1 eq, 6.05 mmol) in Et₂O (25mL) dropwise with stirring. The reaction mixture was stirred at thistemperature for 2 h and then at −55° C. for 4 h, when TLC (hexane:Et₂O;80:20) indicated that the reaction had gone to completion. To thereaction mixture was added a saturated aqueous solution of NH₄Cl (100mL) and allowed to warm to room temperature over 16 h. The reactionmixture was extracted with Et₂O (2×200 mL), and the combined organicphase washed with brine (50 mL), dried (MgSO₄) and evaporated in vacuo.The residue was purified by silica gel column chromatography, elutedwith hexane:Et₂O (80:20) to afford the title compound as a light yellowoil. Yield: 1.37 g (66%). (80% de 2R,3S: 20% de 2S,3S). ¹H-NMR (d₆-DMSO,250 MHz): δ0.0.47 & 0.55 (2×t, J=7.50 & 7.26 Hz —NHCH(CH₂ CH₃)CH(CH₃)OH), 0.99-1.12 (m, 5H, —NHCH(CH₂ CH₃)CH(CH₃ )OH), 2.01 (m, 1H,—NHCH(CH₂CH₃)CH(CH₃)OH), 3.22-3.43 (m, 1H, —NHCH(CH₂CH₃) CH(CH₃)OH),4.41 (d, 1H, J=3.31 Hz, —NHCH(CH₂CH₃)CH(CH₃)OH), 7.14-7.56 (m, 15H,3×Ph).

(3RS, 4R)-4-(Trityl-amino)-hexan-3-ol

To a stirred solution of (R)-2-(trityl-amino)-butyraldehyde (1.5 g, 1eq, 4.53 mmol) in Et₂O (150 mL) under an argon atmosphere at −78° C.,was added ethylmagnesium bromide (3 M in Et₂₀, 1.51 mL, 1 eq, 4.53 mmol)dropwise. The solution was stirred at −78° C. for 2 h, then allowed towarm to room temperature over 16 h. The mixture was recooled to 0° C.,H₂O (150 mL) added, and the organic phase separated. The aqueous phasewas extracted with more Et₂O (2×50 mL), and the combined organic phasewashed with brine (50 mL), dried (MgSO₄) and evaporated in vacuo. Theresidue was purified by silica gel column chromatography, eluted withhexane:ether (90:10) to afford the title compound as a light yellow oil.Yield: 1.13 g (69%). (57% de 3S,4R: 43% de 3R,4R). ¹H-NMR (d₆-DMSO, 250MHz): δ0.45 & 0.69 (t & m, 6H, J=7.43 Hz, —NHCH(CH₂ CH₃ )CH(CH₂ CH₃)OH), 1.12-1.29 (m, 4H, —NHCH (CH₂ CH₃)CH(CH₂ CH₃)OH), 2.16 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 2.54 (m, 1H, —NHCH(CH₂CH₃)CH(CH₂CH₃)OH),3.21-3.40 (m, 1H, —NHCH(CH₂CH₃) CH(CH₂ CH₃)OH), 4.29+4.39 (2×d, 1H,J=4.42 & 5.37 Hz, —NHCH(CH₂CH₃)CH (CH₂CH₃)OH), 7.15-7.52 (m, 15H, 3×Ph).

(3RS, 4S)-4-(Trityl-amino)-hexan-3-ol

To a stirred solution of (R)-2-(trityl-amino)-butyraldehyde (1.5 g, 1eq, 4.53 mmol) in Et₂O (150 mL) under an argon atmosphere at −78° C.,was added ethylmagnesium bromide (3 M in Et₂₀, 1.51 mL, 1 eq, 4.53 mmol)dropwise. The solution was stirred at −78° C. for 2 h, then allowed towarm to room temperature over 16 h. The mixture was recooled to 0° C.,H₂O (150 mL) added, and the organic phase separated. The aqueous phasewas extracted with more Et₂O (2×50 mL), and the combined organic phasewashed with brine (50 mL), dried (MgSO₄) and evaporated in vacuo. Theresidue was purified by silica gel column chromatography, eluted withhexane:ether (90:10) to afford the title compound as a light yellow oil.Yield: 1.19 g (73%). (65% de 3R,4S: 35% de 3S,4S). ¹H-NMR (d₆-DMSO, 250MHz): δ0.46+0.69 (t & m, 6H, J=7.34 Hz, —NHCH(CH₂ CH₃ )CH(CH₂ CH₃ )OH),1.13-1.29 (m, 4H, —NHCH(CH₂ CH₃) CH(CH₂ CH₃)OH), 2.17 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 2.55 (m, 1H, —NHCH(CH₂CH₃)CH(CH₂CH₃)OH),3.20-3.39 (m, 1H, —NHCH(CH₂CH₃)CHCH₂ CH₃)OH), 4.29 & 4.39 (2×d, 1H,J=4.74 & 5.53 Hz, —NHCH(CH₂CH₃)CH (CH₂CH₃)OH), 7.15-7.52 (m, 15H, 3×Ph).

(3RS, 4R)-2-Methyl-4-(trityl-amino)-hexan-3-ol

To a stirred suspension of CuBr.SMe₂ (1.37 g, 2.2 eq, 6.66 mmol) in Et₂O(100 mL) under an argon atmosphere at −78° C., was addedisopropyllithium (0.7 M in pentane, 17.29 mL, 4 eq, 12.1 mmol) dropwise,and the solution allowed to warm to room temperature. The mixture wasrecooled to −70° C., to which was added a solution of(R)-2-(trityl-amino)-butyraldehyde (1 g, 1 eq, 3.03 mmol) in Et₂O (25mL) dropwise with stirring. The reaction mixture was stirred at thistemperature for 1 h, then allowed to warm to −55° C. and stirred at thistemperature for 3 h. To the reaction mixture was added a saturatedaqueous solution of NH₄Cl (100 mL) and allowed to warm to roomtemperature over 16 h. The reaction mixture was extracted with Et₂O(2×200 mL), and the combined organic phase washed with brine (50 mL),dried (MgSO₄) and evaporated in vacuo. The residue was purified bysilica gel gradient column chromatography, eluted with hexane:ether(100:0→90:10) to afford the title compound as a colourless oil. Yield:0.53 g (47%). (50% de 3S,4R: 50% de 3R,4R) ¹H-NMR (d₆-DMSO, 250 MHz): δ0.44 (t, 3H, J=7.03 Hz, —NHCH(CH₂ CH₃ )CH(CH(CH₃)₂)OH), 0.52 & 0.77(2×d, 6H, J=6.48 Hz, —NHCH (CH₂CH₃)CH(CH(CH₃ ) ² )OH), 0.79-1.13 (m, 2H,—NHCH(CH₂ CH₃)CH (CH(CH₃)₂)OH), 1.72 (m, 1H,—NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 2.11 (m, 1H, —NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH),2.77 (m, 1H, —NH CH(CH₂CH₃)CH (CH(CH₃)₂)OH), 2.99 (m, 1H,—NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 4.55 (d, 1H, J=5.21 Hz,—NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 7.15-7.46 (m, 15H, 3×Ph).

(3RS, 4S)-2-Methyl-4-(trityl-amino)-hexan-3-ol

To a stirred suspension of CuBr.SMe₂ (1.37 g, 2.2 eq, 6.66 mmol) in Et₂O(100 mL) under an argon atmosphere at −78° C., was addedisopropyllithium (0.7 M in pentane, 17.29 mL, 4 eq, 12.1 mmol) dropwiseand the solution allowed to warm to room temperature. The mixture wasrecooled to −70° C., to which was added a solution of(S)-2-(trityl-amino)-butyraldehyde (1 g, 1 eq, 3.03 mmol) in Et₂O (25mL) dropwise with stirring. The reaction mixture was stirred at thistemperature for 1 h, then allowed to warm to −55° C. and stirred at thistemperature for 3 h. To the reaction mixture was added a saturatedaqueous solution of NH₄Cl (100 mL) and allowed to warm to roomtemperature over 16 h. The reaction mixture was extracted with Et₂O(2×200 mL), and the combined organic phase washed with brine (50 mL),dried (MgSO₄) and evaporated in vacuo. The residue was purified bysilica gel column chromatography, eluted with hexane:ether (100:0→90:10)to afford the title compound as a colourless oil; Yield: 0.36 g (32%).(50% de 3R,4S: 50% de 3S,4S). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.44 (t, 3H,J=6.79 Hz, —NHCH(CH₂ CH₃ )CH(CH(CH₃)₂)OH), 0.52 & 0.76 (2×d, 6H, J=6.63Hz, —NHCH(CH₂CH₃)CH(CH(CH₃ ) ² )OH), 0.80-1.15 (m, 2H, —NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 1.70 (m, 1H, —NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 2.10 (m,1H, —NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 2.76 (m, 1H,—NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 2.99 (m, 1H, —NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH),4.55 (d, 1H, J=5.84 Hz, —NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 7.17-7.46 (m, 15H,3×Ph).

(3RS, 4R)-2,2-Dimethyl-4-(trityl-amino)-hexan-3-ol

To a stirred suspension of CuBr.SMe₂ (1.37 g, 2.2 eq, 6.66 mmol) in Et₂O(100 mL) under an argon atmosphere at −78° C., was addedtert-butyllithium (1.5 M in pentane, 8.0 mL, 4 eq, 12.0 mmol) dropwiseand the solution allowed to warm to room temperature. The mixture wasrecooled to −55° C., to which was added a solution of(R)-2-(trityl-amino)-butyraldehyde (1 g, 1 eq, 3.03 mmol) in Et₂O (25mL) dropwise with stirring, and stirred at this temperature for 3 h. Tothe reaction mixture was added a saturated aqueous solution of NH₄Cl(100 mL) and allowed to warm to room temperature over 16 h. The reactionmixture was extracted with Et₂O (2×200 mL), and the combined organicphase washed with brine (50 mL), dried (MgSO₄) and evaporated in vacuo.The residue was purified by silica gel gradient column chromatography,eluted with hexane:ether (100:0→90:10) to afford the title compound as alight yellow oil. Yield: 0.57 g (49%). (55% de 3S,4R: 45% de 3R,4R).¹H-NMR (d₆-DMSO, 250 MHz): δ 0.36 & 0.86 (2×t, 3H, J=7.42 Hz, —NH CH(CH₂CH₃ )CH(C(CH₃)₃)OH), 0.57 & 0.71 (2×s, 9H, —NHCH(CH₂CH₃)CH(C(CH₃ )OH),1.38-1.52 (m, 2H, —NHCH (CH₂ CH₃)CH(C(CH₃)₃)OH), 1.99 (m, 1H,—NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 2.27 (m, 1H, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH),2.95 (m, 1H, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 4.22 & 4.77 (2×d, 1H, J=4.425.21 Hz, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 7.14-7.52 (m, 15H, 3×Ph).

(3RS, 4S)-2,2-Dimethyl-4-(trityl-amino)-hexan-3-ol

To a stirred suspension of CuBr.SMe₂ (1.37 g, 2.2 eq, 6.66 mmol) in Et₂O(100 mL) under an argon atmosphere at −78° C., was added tert-butyllithium (1.5 M in pentane, 8.0 mL, 4 eq, 12.0 mmol) dropwise and thesolution allowed to warm to room temperature. The mixture was recooledto −55° C., to which was added a solution of(S)-2-(trityl-amino)-butyraldehyde (1 g, 1 eq, 3.03 mmol) in Et₂O (25mL) dropwise with stirring, and stirred at this temperature for 3 h. Tothe reaction mixture was added a saturated aqueous solution of NH₄Cl(100 mL) and allowed to warm to room temperature over 16 h. The reactionmixture was extracted with Et₂O (2×200 mL), and the combined organicphase washed with brine (50 mL), dried (MgSO₄) and evaporated in vacuo.The residue was purified by silica gel column chromatography, elutedwith hexane: Et₂O (100:0→90:10) to afford the title compound as a lightyellow oil. Yield: 0.47 g (40%). (53% de 3R,4S: 47% de 3S,4S). ¹H-NMR(d₆-DMSO, 250 MHz): δ 0.37 & 0.87 (2×t, 3H, J=7.46 Hz, —NHCH (CH₂ CH₃)CH(C(CH₃)₃)OH), 0.58 & 0.71 (2×s, 9H, —NHCH(CH₂CH₃)CH(C(CH₃ )OH),1.38-1.52 (m, 2H, —NHCH(CH₂ CH₃)CH(C(CH₃)₃)OH), 2.00 (m, 1H,—NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 2.28 (m, 1H, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH),2.95 (m, 1H, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 4.24 & 4.79 (2×d, 1H, J=5.21 &6.16 Hz, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 7.15-7.53 (m, 15H, 3×Ph).

(3R)-3-(Trityl-amino)-pentan-2-one

To a stirred solution of DMSO (2.19 mL, 2.8 eq, 30.86 mmol) in DCM (30mL) under an argon atmosphere at −45° C., was added oxalyl chloride (2 Min DCM, 7.69 mL, 1.4 eq, 15.38 mmol) dropwise. The reaction mixture wasstirred at −45° C. for 1 h, after which time a solution(2S,3R)-3-(trityl-amino)-pentan-2-ol (3.81 g, 1 eq, 11.04 mmol) in DCM(20 mL) was added dropwise with stirring. The reaction mixture wasstirred at this temperature for 4 h, when TLC (hexane:ether; 80:20)indicated that the reaction had gone to completion. To the reactionmixture was added N-ethylpiperidine (7.54 mL, 5 eq, 54.88 mmol), and thesolution allowed to warm to room temperature over 16 h. The reactionmixture was diluted with more DCM (50 mL) and washed with water (200mL). The aqueous phase was extracted with DCM (2×50 mL), and thecombined organic phase washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was dissolved in Et₂O (100 mL), thesolid precipitate removed by filtration and the filtrate was evaporatedin vacuo. The residue was dissolved in hexane (50 mL), the solidprecipitate removed by filtration and the filtrate was evaporated invacuo to afford the title compound as a light yellow oil. Yield: 3.78 g(100%). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.73 (t, 3H, J=7.35 Hz, —NHCH(CH₂CH₃ )C(CH₃)O), 1.47-1.60 (m, 5H, —NHCH(CH₂ CH₃)C(CH₃ )O), 3.12 (d, 1H,J=8.38 Hz, —NHCH(CH₂CH₃)C(CH₃)O), 3.32 (m, 1H, —NHCH(CH₂CH₃)C(CH₃)O),7.16-7.49 (m, 15H, 3×Ph).

(3S)-3-(Trityl-amino)-pentan-2-one

To a stirred solution of DMSO (1.95 mL, 2.8 eq, 27.48 mmol) in DCM (30mL) under an argon atmosphere at −45° C., was added oxalyl chloride (2 Min DCM, 6.85 mL, 1.4 eq, 13.70 mmol) dropwise. The reaction mixture wasstirred at −45° C. for 1 h, after which time a solution(2R,3S)-3-(trityl-amino)-pentan-2-ol (3.39 g, 1 eq, 9.83 mmol) in DCM(20 mL) was added dropwise with stirring. The reaction mixture wasstirred at this temperature for 4 h, when TLC (hexane:ether; 80:20)indicated that the reaction had gone to completion. To the reactionmixture was added N-ethylpiperidine (6.71 mL, 5 eq, 48.84 mmol), and thesolution allowed to warm to room temperature over 16 h. The reactionmixture was diluted with more DCM (50 mL) and washed with water (200mL). The aqueous phase was extracted with DCM (2×50 mL), and thecombined organic phase washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was dissolved in Et₂O (100 mL), thesolid precipitate removed by filtration and the filtrate was evaporatedin vacuo. The residue was dissolved in hexane (50 mL), the solidprecipitate removed by filtration and the filtrate was evaporated invacuo to afford the title compound as a light yellow oil. Yield: 3.15 g(93%). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.73 (t, 3H, J=7.50 Hz, —NHCH(CH₂ CH₃)C(CH₃)O), 1.45-1.62 (m, 5H, —NHCH(CH₂ CH₃)C(CH₃ )O), 3.12 (d, 1H,J=8.53 Hz, —NHCH(CH₂CH₃)C(CH₃)O), 3.31 (m, 1H, —NHCH(CH₂CH₃)C(CH₃)O),7.13-7.45 (m, 15H, 3×Ph).

(3R)-2-Methyl-3-(trityl-amino)-pentan-2-ol

To a stirred solution of (3R)-3-(trityl-amino)-pentan-2-one (0.87 g, 1eq, 2.54 mmol) in Et₂O (100 mL) under an argon atmosphere at roomtemperature, was added methylmagnesium iodide (3 M in ether, 2.54 mL, 3eq, 7.62 mmol) dropwise. The solution was placed in a preheated oil bathat 45° C. and refluxed at this temperature for 16 h. The mixture wasrecooled to 0° C., H₂O (100 mL) added, the solution filtered throughCelite, and the Celite washed with more Et₂O (50 mL). The combinedorganic phase was separated, the aqueous phase was extracted with Et₂O(2×50 mL), and the combined organic phase washed with brine (50 mL),dried (MgSO₄) and evaporated in vacuo. The residue was purified bysilica gel column chromatography, eluted with hexane:ether (100:0→90:10)to afford the title compound as a light yellow oil. Yield: 0.21 g (23%).¹H-NMR (d₆-DMSO, 250 MHz): δ 0.26 (t, J=7.42 Hz, —NHCH(CH₂ CH₃)CH(CH₃)OH), 1.00 & 1.25 (2×s, 6H, —NHCH(CH₂CH₃)C(CH₃ ) ² OH), 0.72-1.43(m, 2H, —NHCH(CH₂ CH₃)C(CH₃)₂OH), 1.84 (m, 1H, —NHCH(CH₂CH₃)C(CH₃)₂ OH),2.90 (m, 1H, —NHCH(CH₂CH₃)C(CH₃)₂OH), 4.32 (s, 1H, —NHCH(CH₂CH₃)C(CH₃)₂OH), 7.17-7.46 (m, 15H, 3×Ph).

(3S)-2-Methyl-3-(trityl-amino)-pentan-2-ol

To a stirred solution of (3S)-3-(trityl-amino)-pentan-2-one (0.59 g, 1eq, 1.72 mmol) in Et₂O (100 mL) under an argon atmosphere at roomtemperature, was added methylmagnesium iodide (3 M in Et₂₀, 1.72 mL, 3eq, 5.16 mmol) dropwise. The solution was placed in a preheated oil bathat 45° C. and refluxed at this temperature for 16 h. The mixture wasrecooled to 0° C., H₂O (100 mL) added, the solution filtered throughCelite, and the Celite washed with more Et₂O (50 mL). The combinedorganic phase was separated, the aqueous phase was extracted with Et₂O(2×50 mL), and the combined organic phase washed with brine (50 mL),dried (MgSO₄) and evaporated in vacuo. The residue was purified bysilica gel column chromatography, eluted with hexane: Et₂O (100:0→90:10)to afford the title compound as a light yellow oil. Yield: 0.10 g (16%).¹H-NMR (d₆-DMSO, 250 MHz): δ0.27 (t, J=7.10 Hz, —NHCH(CH₂ CH₃)CH(CH₃)OH), 0.99 & 1.25 (2×s, 6H, —NHCH(CH₂CH₃)C(CH₃ ) ² OH), 0.75-1.42(m, 2H, —NHCH(CH₂ CH₃)C(CH₃)₂OH), 1.88 (m, 1H, —NHCH(CH₂CH₃)C(CH₃)₂ OH),2.92 (m, 1H, —NHCH(CH₂CH₃)C(CH₃)₂OH), 4.32 (s, 1H,—NHCH(CH₂CH₃)C(CH₃)₂OH), 7.18-7.46 (m, 15H, 3×Ph).

(2S,3R)-3-Amino-pentan-2-ol

To a stirred solution of (2S,3R)-3-(trityl-amino)-pentan-2-ol (1.32 g, 1eq, 3.83 mmol) in DCM (50 mL) under an argon atmosphere at roomtemperature, was added CF₃COOH (10 mL) dropwise, and the solution wasstirred at this temperature for 1 h. The solvent was evaporated in vacuoand the residue was precipitated from Et₂O (15 mL) with hexane (300 mL)with stirring to give a yellow oil. The solvent was decanted from theoil, and the oil was washed with hexane (30 mL) and dried in vacuo toafford the title compound as a light yellow oil. Yield: 0.30 g (99%).(80% de 2S,3R: 20% de 2R,3R). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.915 & 0.924(2×t, 3H, J=7.50 & 7.58 Hz, NH₂CH(CH₂ CH₃ )CH(CH₃)OH), 1.06 & 1.13 (2×d,J=6.48 & 6.32 Hz), NH₂CH (CH₂CH₃)CH(CH₃ )OH), 1.41-1.59 (m, 2H,NH₂CH(CH₂ CH₃)CH(CH₃)OH), 2.77 & 2.93 (2×m, 1H, NH₂CH(CH₂CH₃)CH(CH₃)OH), 3.62-3.72 & 3.80-3.90 (2×m, 1H,NH₂CH(CH₂CH₃)CH(CH₃)OH), 7.75 (bs, 2H, NH₂ ).

(2R,3S)-3-Amino-pentan-2-ol

To a stirred solution of (2R,3S)-3-(trityl-amino)-pentan-2-ol (1.64 g, 1eq, 4.75 mmol) in DCM (50 mL) under an argon atmosphere at roomtemperature, was added CF₃COOH (10 mL) dropwise, and the solution wasstirred at this temperature for 1 h. The solvent was evaporated in vacuoand the residue was precipitated from Et₂O (15 mL) with hexane (300 mL)with stirring to give a yellow oil. The solvent was decanted from theoil, and the oil was washed with hexane (30 mL) and dried in vacuo toafford the title compound as a light yellow oil. Yield: 0.30 g (98%).(80% de 2R,3S: 20% de 2S,3S). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.913 & 0.923(2×t, 3H, J=7.50 & 7.50 Hz, NH₂CH(CH₂ CH₃ )CH(CH₃)OH), 1.11 & 1.18 (2×d,J=6.48 & 6.48 Hz), NH₂CH(CH₂CH₃)CH(CH₃ )OH), 1.41-1.65 (m, 2H, NH₂CH(CH₂CH₃)CH(CH₃)OH), 2.76+2.93 (2×m, 1H, NH₂ CH(CH₂CH₃)CH(CH₃)OH), 3.61-3.69& 3.80-3.90 (2×m, 1H, NH₂CH(CH₂CH₃)CH(CH₃)OH), 7.73 (bs, 2H, NH₂ ).

(3RS, 4R)-4-Amino-hexan-3-ol

To a stirred solution of (3RS,4R)-4-(trityl-amino)-hexan-3-ol (1.13 g, 1eq, 3.14 mmol) in DCM (15 mL) under an argon atmosphere at roomtemperature, was added CF₃COOH (7 mL) dropwise, and the solution wasstirred at this temperature for 4 h. The solvent was evaporated invacuo, EtOH (20 mL) added, and removed in vacuo, and this processrepeated twice. The residue was precipitated from Et₂O (5 mL) withhexane (40 mL) with stirring to give a yellow oil. The solvent wasdecanted from the oil, and the oil was washed with hexane (30 mL) anddried in vacuo to afford the title compound as a light yellow oil.Yield: 0.37 g (100%). (57% de 3S,4R: 43% de 3R,4R). ¹H-NMR (d₆-DMSO, 250MHz): δ0.79 & 0.92 (t & m, 6H, J=7.42 Hz, NH₂CH(CH₂ CH₃ )CH(CH₂ CH₃)OH), 1.30-1.67 (m, 4H, NH₂CH(CH₂ CH₃)CH(CH₂ CH₃)OH), 2.70 (m, 1H,NH₂CH(CH₂CH₃)CH(CH₂CH₃)OH), 2.84 & 2.96 (2×m, 1H, NH₂CH(CH₂CH₃)CH(CH₂CH₃)OH), 3.41 & 3.56 (2×m, 1H,NH₂CH(CH₂CH₃)CH(CH₂CH₃)OH), 7.71 (bs, 2H, NH₂ CH(CH₂CH₃)CH(CH₂CH₃)OH).

(3RS, 4S)-4-Amino-hexan-3-ol

To a stirred solution of (3RS,4S)-4-(trityl-amino)-hexan-3-ol (1.19 g, 1eq, 3.31 mmol) in DCM (15 mL) under an argon atmosphere at roomtemperature, was added CF₃COOH (7 mL) dropwise, and the solution wasstirred at this temperature for 4 h. The solvent was evaporated invacuo, EtOH (20 mL) added, and removed in vacuo, and this processrepeated twice. The residue was precipitated from Et₂O (5 mL) withhexane (40 mL) with stirring to give a yellow oil. The solvent wasdecanted from the oil, and the oil was washed with hexane (30 mL) anddried in vacuo to afford the title compound. Yield: 0.39 g (99%). (65%de 3R,4S: 35% de 3S,4S). ¹H-NMR (d₆-DMSO, 250 MHz): δ 0.79 & 0.92 (t &m, 6H, J=7.50 Hz, NH₂CH(CH₂ CH₃ )CH(CH₂ CH₃ )OH), 1.22-1.68 (m, 4H,NH₂CH(CH₂ CH₃)CH(CH₂ CH₃)OH), 2.71 (m, 1H, NH₂CH(CH₂CH₃)CH(CH₂CH₃)OH),2.83 & 2.95 (2×m, 1H, NH₂ CH(CH₂CH₃)CH(CH₂CH₃)OH), 3.39 & 3.54 (2×m, 1H,NH₂CH(CH₂CH₃)CH(CH₂CH₃)OH), 7.77 (bs, 2H, NH₂ CH(CH₂CH₃)CH(CH₂CH₃)OH).

(3RS, 4R)-4-Amino-2-methyl-hexan-3-ol

To a stirred solution of (3RS,4R)-2-methyl-4-(trityl-amino)-hexan-3-ol(0.53 g, 1 eq, 1.41 mmol) in DCM (20 mL) under an argon atmosphere atroom temperature, was added CF₃COOH (5 mL) dropwise, and the solutionwas stirred at this temperature for 1 h. The solvent was evaporated invacuo, the residue was precipitated from Et₂O (10 mL) with hexane (90mL) with stirring to give a yellow oil. The solvent was decanted fromthe oil, and the oil was washed with hexane (20 mL) and dried in vacuoto afford the title compound as a light yellow oil. Yield: 0.18 g(100%). (50% de 3S,4R: 50% de 3R,4R). ¹H-NMR (d₆-DMSO, 250 MHz):δ0.85-0.99 (m, 9H, NH₂CH(CH₂ CH₃ )CH(CH(CH₃)₂)OH), 1.42-1.79 (m, 2H,NH₂CH(CH₂ CH₃)CH(CH(CH₃)₂)OH), 2.95 (m, 1H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH), 3.18 (m, 1H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH),3.37 (m, 1H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH), 7.58 (bs, 2H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH).

(3RS, 4S)-4-Amino-2-methyl-hexan-3-ol

To a stirred solution of (3RS,4S)-2-methyl-4-(trityl-amino)-hexan-3-ol(0.36 g, 1 eq, 0.97 mmol) in DCM (20 mL) under an argon atmosphere atroom temperature, was added CF₃COOH (5 mL) dropwise, and the solutionwas stirred at this temperature for 1 h. The solvent was evaporated invacuo, the residue was precipitated from Et₂O (10 mL) with hexane (90mL) with stirring to give a yellow oil. The solvent was decanted fromthe oil, and the oil was washed with hexane (20 mL) and dried in vacuoto afford the title compound as a light yellow oil. Yield: 0.13 g(100%). (50% de 3R,4S: 50% de 3S,4S) ¹H-NMR (d₆-DMSO, 250 MHz): δ0.85-1.01 (m, 9H, NH₂CH (CH₂ CH₃ )CH(CH(CH₃ )₂)OH), 1.44-1.76 (m, 2H,NH₂CH(CH₂ CH₃)CH(CH(CH₃)₂)OH), 2.94 (m, 1H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH), 3.17 (m, 1H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH),3.40 (m, 1H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH), 7.54 (bs, 2H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH).

(3RS, 4R)-4-Amino-2,2-dimethyl-hexan-3-ol

To a stirred solution of(3RS,4R)-2,2-dimethyl-4-(trityl-amino)-hexan-3-ol (0.57 g, 1 eq, 1.47mmol) in DCM (10 mL) under an argon atmosphere at room temperature, wasadded CF₃COOH (5 mL) dropwise, and the solution was stirred at thistemperature for 1 h. The solvent was evaporated in vacuo, the residuewas precipitated from Et₂O (3 mL) with hexane (20 mL) with stirring togive a yellow oil. The solvent was decanted from the oil, and the oilwas washed with hexane (20 mL) and dried in vacuo to afford the titlecompound as a light yellow oil. Yield: 0.21 g (100%). (55% de 3S,4R: 45%de 3R,4R). ¹H-NMR (d₆-DMSO, 250 MHz): δ 0.84-0.99 (m, 3H, NH₂CH(CH₂ CH₃)CH(C(CH₃)₃)OH), 1.25-1.29 (m, 9H, NH₂CH(CH₂CH₃)CH(C(CH₃ ) ³ )OH),1.20-1.72 (m, 2H, NH₂CH(CH₂ CH₃)CH(C(CH₃)₃)OH), 3.14 (m, 1H, NH₂CH(CH₂CH₃)CH(C(CH₃)₃)OH), 3.39 (m, 1H, NH₂CH(CH₂CH₃)CH(C(CH₃)₃)OH), 3.65(m, 1H, NH₂CH(CH₂CH₃)CH(C(CH₃)₃)OH), 7.43, 7.77 & 8.54 (3×bs, 2H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH).

(3RS, 4S)-4-Amino-2,2-dimethyl-hexan-3-ol

To a stirred solution of(3RS,4S)-2,2-dimethyl-4-(trityl-amino)-hexan-3-ol (0.47 g, 1 eq, 1.21mmol) in DCM (10 mL) under an argon atmosphere at room temperature, wasadded CF₃COOH (5 mL) dropwise, and the solution was stirred at thistemperature for 1 h. The solvent was evaporated in vacuo, the residuewas precipitated from Et₂O (3 mL) with hexane (20 mL) with stirring togive a yellow oil. The solvent was decanted from the oil, and the oilwas washed with hexane (20 mL) and dried in vacuo to afford the titlecompound as a light yellow oil. Yield: 0.18 g (99%). (53% de 3R,4S: 47%de 3S,4S). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.86-0.99 (m, 3H, NH₂CH(CH₂ CH₃)CH(C(CH₃)₃)OH), 1.25-1.30 (m, 9H, NH₂CH(CH₂CH₃)CH(C(CH₃ )₃)OH),1.20-1.67 (m, 2H, NH₂CH(CH₂ CH₃)CH(C(CH₃)₃)OH), 3.14 (m, 1H, NH₂CH(CH₂CH₃)CH(C(CH₃)₃)OH), 3.38 (m, 1H, NH₂CH(CH₂CH₃)CH(C(CH₃)₃)OH), 3.64(m, 1H, NH₂CH(CH₂CH₃)CH(C(CH₃)₃)OH), 7.41, 7.73 & 8.44 (3×bs, 2H, NH₂CH(CH₂CH₃)CH(CH(CH₃)₂)OH).

(3R)-3-Amino-2-methyl-pentan-2-ol

To a stirred solution of (3R)-2-methyl-3-(trityl-amino)-pentan-2-ol(0.21 g, 1 eq, 0.60 mmol) in DCM (5 mL) under an argon atmosphere atroom temperature, was added CF₃COOH (2.5 mL) dropwise, and the solutionwas stirred at this temperature for 1 h. The solvent was evaporated invacuo and the residue was precipitated from Et₂O (15 mL) with hexane(300 mL) with stirring to give a yellow oil. The solvent was decantedfrom the oil, and the oil was washed with hexane (30 mL) and dried invacuo to afford the title compound as a light yellow oil; Yield: 0.07 g(100%). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.97 (t, 3H, J=7.42 Hz, NH₂CH(CH₂CH₃ )C(CH₃)₂OH), 1.06 & 1.19 (2×s, 6H, NH₂CH(CH₂CH₃)C(CH₃ )₂OH),1.28-1.71 (m, 2H, NH₂CH(CH₂ CH₃)C(CH₃)₂OH), 2.72 (m, 1H, NH₂CH(CH₂CH₃)C(CH₃)₂OH), 5.21 (s, 1H, NH₂CH (CH₂CH₃)C(CH₃)₂ OH), 7.63 (bs,2H, NH₂ CH(CH₂CH₃)C(CH₃)₂OH).

(3S)-3-Amino-2-methyl-pentan-2-ol

To a stirred solution of (3S)-2-methyl-3-(trityl-amino)-pentan-2-ol(0.38 g, 1 eq, 1.06 mmol) in DCM (5 mL) under an argon atmosphere atroom temperature, was added CF₃COOH (2.5 mL) dropwise, and the solutionwas stirred at this temperature for 1 h. The solvent was evaporated invacuo and the residue was precipitated from Et₂O (15 mL) with hexane(300 mL) with stirring to give a yellow oil. The solvent was decantedfrom the oil, and the oil was washed with hexane (30 mL) and dried invacuo to afford the title compound as a light yellow oil. Yield: 0.12 g(99%). ¹H-NMR (d₆-DMSO, 250 MHz): δ0.97 (t, 3H, J=7.42 Hz, NH₂CH(CH₂ CH₃)C(CH₃)₂OH), 1.07 & 1.19 (2×s, 6H, NH₂CH(CH₂CH₃)C(CH₃ )₂OH), 1.28-1.61(m, 2H, NH₂CH (CH₂ CH₃)C(CH₃)₂OH), 2.72 (m, 1H, NH₂CH(CH₂CH₃)C(CH₃)₂OH), 5.21 (s, 1H, NH₂CH(CH₂CH₃)C(CH₃)₂ OH), 7.63 (bs,2H, NH₂ CH(CH₂CH₃)C(CH₃)₂OH).

6-Chloro-2-fluoro-9H-purine

This compound was prepared by a modification of a literature procedures(Gray, N. S.; Kwon, S.; Schultz, P. G. Tetrahedron Lett. 1997, 38(7),1161-1164.)

6-Chloro-9H-purin-2-ylamine (75.0 g, 0.44 mol) was suspended in aq HBF₄(1.5 L of 48% w/w solution in H₂O). This mixture was cooled to −15° C.and was stirred vigorously. NaNO₂ (2.5 L of an 0.3 M aq solution) wasthen added slowly over 75 min with stirring and careful temperaturecontrol (<10° C.). After complete addition, the pale yellow solution wasfurther stirred at room temperature for 30 min. It was then re-cooled to−15° C. and was neutralised carefully to pH=6.2 with NaOH (50% w/v aqsolution). This solution was rotary evaporated to semi-dryness. Theresulting cake was divided with a spatula and dried under high vacuumovernight. The resulting yellow powder was dry-loaded onto a flashchromatography column (24×15 cm SiO₂ bed), which was eluted withCH₂Cl₂/MeOH, 9:1. Appropriate fractions were collected, pooled, andevaporated. After drying in vacuo, the title compound (34.8 g, 48%) wasobtained as a colourless powder. TLC: R_(f)=0.25 (CH₂Cl₂/MeOH, 9:1),starting material R_(f)=0.16. m/z 173 (MH⁺, 100), 175 (MH⁺², 33).

(2-Fluoro-9H-purin-6-yl)-pyridin-3-ylmethyl-amine

To a stirred solution of 6-chloro-2-fluoropurine (0.9 g, 1 eq, 5.22mmol) in n-BuOH (60 mL) under an argon atmosphere, cooled to −20° C.,was added DIEA (2.5 mL, 2.75 eq, 14.35 mmol) followed byC-pyridin-3-yl-methylamine (0.58 mL, 1.1 eq, 5.69 mmol). The reactionmixture was stirred at −20° C. for 3 h, and then allowed to return toroom temperature over 2 h, when TLC (CHCl₃: MeOH; 90:10) indicated thatthe reaction had gone to completion. The solvent was evaporated in vacuoand the residue was purified by gradient column chromatography on silicagel, eluted with CHCl₃:MeOH (97:3→90:10), to afford the title compoundas a white solid. Yield: 1.08 g (85%). Mp 218-220° C. ¹H-NMR (d₆-DMSO,250 MHz): δ4.65 (d, 2H, J=5.37 Hz, —HNCH₂ -Pyr), 7.34, 8.43, 8.57 (3×m,4H, Pyr), 8.10 (s, 1H, —N═CH—NH—), 8.83 (bs, 1H, —HNCH₂-Pyr), 13.07 (bs,1H, —N═CH—NH—). FABMS m/z (relative intensity): 245 ([M+H]⁺, 40), 176(27), 154 (100), 136 (74). Accurate Mass (M+H): Actual: 245.0951,Measured: 245.0942. Microanalysis (Expected: Measured) C₁₁H₉N₆F .0.2H₂O:C, 53.31; 54.03; H, 3.82; 3.83; N, 33.91; 32.74.

(2-Fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine

To a stirred solution of(2-fluoro-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (1.00 g, 1 eq, 4.10mmol) in DMA (12 mL) under an argon atmosphere, at RT, was added K₂CO₃(powdered, anhydrous, 2.75 g, 4.85 eq, 19.90 mmol) followed2-bromopropane (3.75 mL, 9.75 eq, 39.93 mmol). The reaction mixture wasstirred at RT for 48 h, when TLC (CHCl₃:MeOH; 90:10) indicated that thereaction had gone to completion. The solvent was evaporated in vacuo andthe residue partitioned between water (200 mL) and EtOAc (100 mL), theaqueous phase was separated and extracted with more EtOAc (2×50 mL). Thebulked organic phase was washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo, and the residue was purified by gradient columnchromatography on silica gel, eluted with CHCl₃:MeOH (100:0→95:5), toafford the title compound as a white solid. Yield: 0.73 g (62%). Mp131-133° C. ¹H-NMR (d₆-DMSO, 250 MHz): δ1.49 (2×s, 6H, —CH(CH₃ )₂), 4.63(m, 3H, —CH(CH₃)₂ & —HNCH₂ -Pyr), 7.34, 7.24, 8.44, 8.58 (4×m, 4H, Pyr),8.26 (s, 1H, —N═CH—N—), 8.95 (bs, 1H, —HNCH₂-Pyr). FABMS m/z (relativeintensity): 287 ([M+H]⁺, 100), 245 (8), 154 (23), 136 (18). AccurateMass (M+H): Actual: 287.1420, Measured: 287.1412. Microanalysis(Expected: Measured) C₁₄H₁₅N₆F: C, 58.73; 58.57; H, 5.28; 5.21; N,29.35; 29.27.

(2S3R)-3-{(9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (30 mg, 1eq, 0.10 mmol) in n-BuOH/DMSO (2.5 mL, 4:1) at room temperature under anargon atmosphere was added DIEA (0.2 mL, 10.96 eq, 1.14 mmol) followedby (2S,3R)-3-amino-pentan-2-ol (60 mg, 5.5 eq, 0.58 mmol). The reactionmixture was placed in a preheated oil bath at 160° C. and stirred atthis temperature for 72 h. The reaction mixture was allowed to cool toroom temperature and the solvent was evaporated in vacuo. The residuewas partitioned between EtOAc (50 mL) and water (50 mL), the aqueousphase was extracted with more EtOAc (2×25 mL), and the combined organicphase was washed with brine (50 mL), dried (MgSO₄) and evaporated invacuo. The residue was purified by gradient column chromatography onsilica gel eluted with CHCl₃:MeOH (100:0→98:2), to afford the titlecompound as a white solid. Yield: 22 mg (57%). (80% de 3R,2S: 20% de3R,2R). ¹H-NMR (d-CDCl₃, 250 MHz): δ 0.90, 1.05 (2×t, 3H, J=6.95 & 7.42Hz, —NHCH (CH₂ CH₃ )CH(CH₃)OH), 1.17 & 1.25 (2×d, 3H, J=6.32 & 6.16 Hz,—NHCH (CH₂CH₃)CH(CH₃ )OH) 1.57 (d, 6H, J=6.79 Hz, —CH(CH₃ ) ² ),1.77-2.09 (m, 2H, —NHCH(CH₂ CH₃)CH(CH₃)OH), 3.91-4.03 (m, 2H,—NHCH(CH₂CH₃)CH(CH₃)OH), 4.58-4.69 (m, 1H, —CH(CH₃)₂), 4.83-5.00 (m, 3H,—HNCH₂ -Pyr & OH), 6.16 (m, 1H, —NHCH(CH₂CH₃)CH(CH₃)OH), 7.24-7.33 (m,3H, 2×Pyr-H & —N═CH—N—), 7.55 (m, 1H, Pyr-H), 7.74 (d, 1H, J=7.42 Hz,Pyr-H), 8.67 (m, 1H, HNCH₂-Pyr). FABMS m/z (relative intensity): 370([M+H]⁺, 100), 324 (35), 289 (37), 243 (65), 199 (85). Accurate Mass(M+H): Actual: 370.2355, Measured: 370.2347.

(2R3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (30 mg, 1eq, 0.10 mmol) in n-BuOH/DMSO (2.5 mL, 4:1) at room temperature under anargon atmosphere was added DIEA (0.2 mL, 10.96 eq, 1.14 mmol) followedby (2R,3S)-3-amino-pentan-2-ol (60 mg, 5.5 eq, 0.58 mmol). The reactionmixture was placed in a preheated oil bath at 160° C. and stirred atthis temperature for 72 h. The reaction mixture was allowed to cool toroom temperature and the solvent was evaporated in vacuo. The residuewas partitioned between EtOAc (50 mL) and water (50 mL), the aqueousphase was extracted with more EtOAc (2×25 mL), and the combined organicphase was washed with brine (50 mL), dried (MgSO₄) and evaporated invacuo. The residue was purified by gradient column chromatography onsilica gel eluted with CHCl₃:MeOH (100:0→98:2), to afford the titlecompound as a white solid. Yield: 25 mg (65%). (80% de 3S,2R: 20% de3S,2S). ¹H-NMR (d-CDCl₃, 250 MHz): δ0.90 & 1.05 (2×t, 3H, J=6.48 & 7.42Hz, —NHCH(CH₂ CH₃ ) CH(CH₃)OH), 1.16 & 1.24 (2×d, 3H, J=6.31 & 6.16 Hz,—NHCH(CH₂CH₃) CH(CH₃ )OH) 1.57 (d, 6H, J=6.79 Hz, —CH(CH₃ ) ² ),1.77-2.09 (m, 2H, —NHCH(CH₂ CH₃)CH(CH₃)OH), 3.91-4.04 (m, 2H,—NHCH(CH₂CH₃)CH(CH₃)OH), 4.58-4.69 (m, 1H, —CH(CH₃)₂), 4.83 (m, 3H,—HNCH₂ -Pyr & OH), 6.11-6.23 (m, 1H, —NHCH(CH₂CH₃)CH(CH₃)OH), 7.24-7.32(m, 3H, 2×Pyr-H+ —N═CH—N—), 7.53-7.57 (m, 1H, Pyr-H), 7.74 (d, 1H,J=7.58 Hz, Pyr-H), 8.67 (m, 1H, HNCH₂-Pyr). FABMS m/z (relativeintensity): 370 ([M+H]⁺, 100), 324 (40), 289 (15), 243 (10), 233 (13),199 (10). Accurate Mass (M+H): Actual: 370.2355, Measured: 370.2347.

(3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-3-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (20 mg, 1eq, 0.07 mmol) in n-BuOH/DMSO (3.75 mL, 4:1) at room temperature underan argon atmosphere was added DIEA (0.18 mL, 15 eq, 1.03 mmol) followedby (3RS,4R)-4-amino-hexan-3-ol (110 mg, 13 eq, 0.94 mmol). The reactionmixture was placed in a preheated oil bath at 140° C. and stirred atthis temperature for 72 h. The reaction mixture was allowed to cool toroom temperature and the solvent was evaporated in vacuo. The residuewas partitioned between EtOAc (50 mL) and brine/water (1:1, 100 mL), theaqueous phase was extracted with more EtOAc (2×25 mL), and the combinedorganic phase was washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was purified by gradient columnchromatography on silica gel eluted with CHCl₃:MeOH (100:0→98:2), toafford the title compound as a white solid. Yield: 23 mg (75%). (57% de4R,3S: 43% de 4R,3R). ¹H-NMR (d-CDCl₃, 250 MHz): δ 0.87-1.07 (m, 6H,—NHCH(CH₂ CH₃ ) CH(CH₂ CH₃ )OH), 1.56 (d, 6H, J=6.63 Hz, & —CH(CH₃ ) ²), 1.43-1.63 (m, 4H, —NHCH(CH₂ CH₃)CH(CH₂ CH₃)OH), 3.44 (d, 1H, J=6.31Hz, OH), 3.58-3.71 (m, 1H, —NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 3.89-4.01 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 4.56-4.70 (m, 1H, —CH(CH₃)₂), 4.76-4.95 (m,2H, —HNCH₂ -Pyr), 5.20-5.29 & 6.17-6.34 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 7.19-7.38 (m, 3H, 2×Pyr-H & —N═CH—N—),7.48-7.60 (m, 1H, Pyr-H), 7.72 (d, 1H, J=7.74 Hz, Pyr-H), 8.67 (m, 1H,HNCH₂-Pyr). FABMS m/z (relative intensity): 384 ([M+H]⁺, 100), 324 (50),297 (30). Accurate Mass (M+H): Actual: 384.2512, Measured: 384.2500.

(3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-3-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (20 mg, 1eq, 0.07 mmol) in n-BuOH/DMSO (3.75 mL, 4:1) at room temperature underan argon atmosphere was added DIEA (0.18 mL, 15 eq, 1.03 mmol) followedby (3RS,4S)-4-amino-hexan-3-ol (110 mg, 13 eq, 0.94 mmol). The reactionmixture was placed in a preheated oil bath at 140° C. and stirred atthis temperature for 72 h. The reaction mixture was allowed to cool toroom temperature and the solvent was evaporated in vacuo. The residuewas partitioned between EtOAc (50 mL) and brine/water (1:1, 100 mL), theaqueous phase was extracted with more EtOAc (2×25 mL), and the combinedorganic phase was washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was purified by gradient columnchromatography on silica gel eluted with CHCl₃:MeOH (100:0→98:2), toafford the title compound as a white solid. Yield: 15.5 mg (58%). (57%de 4S,3R: 43% de 4S,3S). ¹H-NMR (d-CDCl₃, 250 MHz): δ 0.84-1.09 (m, 6H,—NHCH(CH₂ CH₃ ) CH(CH₂ CH₃ )OH), 1.57 (d, 6H, J=6.48 Hz, & —CH(CH₃ ) ²), 1.42-1.64 (m, 4H, —NHCH(CH₂ CH₃)CH(CH₂ CH₃)OH), 3.45 (d, 1H, J=6.31Hz, OH), 3.55-3.71 (m, 1H, —NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 3.88-4.03 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 4.56-4.74 (m, 1H, —CH(CH₃)₂), 4.78-4.99 (m,2H, —HNCH₂ -Pyr), 5.20-5.29 & 6.20-6.39 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 7.20-7.36 (m, 3H, 2×Pyr-H+ —N═CH—N—),7.49-7.60 (m, 1H, Pyr-H), 7.72 (d, 1H, J=7.82 Hz, Pyr-H), 8.71 (m, 1H,HNCH₂-Pyr). FABMS m/z (relative intensity): 384 ([M+H]⁺, 100), 324 (35),297 (15). Accurate Mass (M+H): Actual: 384.2512, Measured: 384.2500.

(3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-hexan-3-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (30 mg, 1eq, 0.10 mmol) in n-BuOH/DMSO (2.5 mL, 4:1) at room temperature under anargon atmosphere was added DIEA (0.10 mL, 5.5 eq, 0.57 mmol) followed by(3RS,4R)-4-amino-2-methyl-hexan-3-ol (42 mg, 3.0 eq, 0.32 mmol). Thereaction mixture was placed in a preheated oil bath at 140° C. andstirred at this temperature for 72 h. The reaction mixture was allowedto cool to room temperature and the solvent was evaporated in vacuo. Theresidue was partitioned between EtOAc (50 mL) and brine/water (1:1, 100mL), the aqueous phase was extracted with more EtOAc (2×25 mL), and thecombined organic phase was washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was purified by gradient columnchromatography on silica gel eluted with CHCl₃:MeOH (100:0→98:2), toafford the title compound as a white solid. Yield: 6.3 mg (15%). 50% de4R,3S: 50% de 4R,3R). ¹H-NMR (d-CDCl₃, 250 MHz): δ 0.93-1.04 (m, 9H,—NHCH(CH₂ CH₃ )CH(CH(CH₃ ) ² )OH), 1.57 (d, 6H, J=6.95 Hz, —CH(CH₃ ) ²), 1.65-1.89 (m, 4H, —NHCH(CH₂ CH₃)CH(CH(CH₃)₂)OH), 3.22-3.32 (m, 1H,—NHCH(CH₂CH₃)CH (CH(CH₃)₂)OH), 3.82-3.97 (m, 1H,—NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 4.54-4.70 (m, 1H, —CH(CH₃)₂), 4.78-4.88(m, 2H, —HNCH₂ -Pyr), 5.03-5.14 & 6.08-6.20 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 7.22-7.36 (m, 3H, 2×Pyr-H & —N═CH—N—),7.49-7.58 (m, 1H, Pyr-H), 7.71 (d, 1H, J=7.90 Hz, Pyr-H), 8.47-8.73 (m,1H, HNCH₂-Pyr). FABMS m/z (relative intensity): 398 ([M+H]⁺, 100), 324(50). Accurate Mass (M+H): Actual: 398.2668, Measured: 398.2654.

(3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-hexan-3-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (30 mg, 1eq, 0.10 mmol) in n-BuOH/DMSO (2.5 mL, 4:1) at room temperature under anargon atmosphere was added DIEA (0.20 mL, 11 eq, 1.14 mmol) followed by(3RS,4S)-4-amino-2-methyl-hexan-3-ol (28 mg, 2.0 eq, 0.21 mmol). Thereaction mixture was placed in a preheated oil bath at 160° C. andstirred at this temperature for 72 h. The reaction mixture was allowedto cool to room temperature and the solvent was evaporated in vacuo. Theresidue was partitioned between EtOAc (50 mL) and brine/water (1:1, 100mL), the aqueous phase was extracted with more EtOAc (2×25 mL), and thecombined organic phase was washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was purified by gradient columnchromatography on silica gel eluted with CHCl₃:MeOH (100:0→98:2), toafford the title compound as a white solid. Yield: 4.3 mg (10%). 50% de4S,3R: 50% de 4S,3S). ¹H-NMR (d-CDCl₃, 250 MHz): δ 0.91-1.05 (m, 9H,—NHCH(CH₂ CH₃ )CH(CH(CH₃ ) ² )OH), 1.57 (d, 6H, J=6.95 Hz, —CH(CH₃ ) ²), 1.60-1.95 (m, 4H, —NHCH(CH₂ CH₃)CH(CH(CH₃)₂)OH), 3.23-3.33 (m, 1H,—NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 3.84-4.04 (m, 1H,—NHCH(CH₂CH₃)CH(CH(CH₃)₂)OH), 4.56-4.70 (m, 1H, —CH(CH₃)₂), 4.77-4.95(m, 2H, —HNCH₂ -Pyr), 5.26-5.43+6.27-6.50 (m, 1H,—NHCH(CH₂CH₃)CH(CH₂CH₃)OH), 7.23-7.37 (m, 3H, 2×Pyr-H & —N═CH—N—),7.51-7.63 (m, 1H, Pyr-H), 7.73 (d, 1H, J=7.74 Hz, Pyr-H), 8.48-8.76 (m,1H, HNCH₂-Pyr). FABMS m/z (relative intensity): 398 ([M+H]⁺, 100), 324(60). Accurate Mass (M+H): Actual: 398.2668, Measured: 398.2654.

(3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (30 mg, 1eq, 0.10 mmol) in n-BuOH/DMSO (5 mL, 4:1) at room temperature under anargon atmosphere was added DIEA (0.10 mL, 5.5 eq, 0.57 mmol) followed by(3RS,4R)-4-amino-2,2-dimethyl-hexan-3-ol (52 mg, 3.41 eq, 0.36 mmol).The reaction mixture was placed in a preheated oil bath at 140° C. andstirred at this temperature for 72 h. The reaction mixture was allowedto cool to room temperature and the solvent was evaporated in vacuo. Theresidue was partitioned between EtOAc (50 mL) and brine/water (1:1, 100mL), the aqueous phase was extracted with more EtOAc (2×25 mL), and thecombined organic phase was washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was purified by gradient columnchromatography on silica gel eluted with CHCl₃:MeOH (100:0→98:2), toafford the title compound as a white solid. Yield: 7.3 mg (17%). (55% de4R,3S: 45% de 4R,3R). ¹H-NMR (d-CDCl₃, 250 MHz): δ 0.99-1.03 (m, 12H,—NHCH(CH₂ CH₃ )CH(C(CH₃ ) ³ ), 1.56 & 1.58 (2×d, 6H, J=6.63 & 6.79 Hz,—CH(CH₃ ) ² ), 1.69-1.91 (m, 2H, —NHCH(CH₂ CH₃)CH(C(CH₃)₃)OH), 3.55 (d,1H, J=1.74 Hz, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 3.76-3.87 (m, 1H,—NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 4.57-4.70 (m, 1H, —CH(CH₃)₂), 4.80-4.89 (m,2H, —HNCH₂ -Pyr), 5.22-5.35 & 6.07-6.23 (m, 1H,—NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 7.24-7.36 (m, 3H, 2×Pyr-H & —N═CH—N—), 7.55(s, 1H, Pyr-H), 7.74 (d, 1H, J=7.58 Hz, Pyr-H), 8.50-8.74 (m, 1H,HNCH₂-Pyr). FABMS m/z (relative intensity): 412 ([M+H]⁺, 100), 324 (80).Accurate Mass (M+H): Actual: 412.2825, Measured: 412.2835.

(3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol

To a stirred solution of(3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol(30 mg, 1 eq, 0.10 mmol) in n-BuOH/DMSO (5 mL, 4:1) at room temperatureunder an argon atmosphere was added DIEA (0.10 mL, 5.5 eq, 0.57 mmol)followed by (3RS,4S)-4-amino-2,2-dimethyl-hexan-3-ol (43 mg, 2.81 eq,0.29 mmol). The reaction mixture was placed in a preheated oil bath at140° C. and stirred at this temperature for 72 h. The reaction mixturewas allowed to cool to room temperature and the solvent was evaporatedin vacuo. The residue was partitioned between EtOAc (50 mL) andbrine/water (1:1, 100 mL), the aqueous phase was extracted with moreEtOAc (2×25 mL), and the combined organic phase was washed with brine(50 mL), dried (MgSO₄) and evaporated in vacuo. The residue was purifiedby gradient column chromatography on silica gel eluted with CHCl₃:MeOH(100:0→98:2), to afford the title compound as a white solid. Yield: 5.4mg (13%). (53% de 4S,3R: 47% de 4S,3S). ¹H-NMR (d-CDCl₃, 250 MHz):δ0.99-1.03 (m, 12H, —NHCH(CH₂ CH₃ )CH(C(CH₃ ) ³ )OH), 1.57 & 1.59 (2×d,6H, J=6.79 & 6.79 Hz, —CH(CH₃ ) ² ), 1.70-1.93 (m, 2H, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 3.55 (d, 1H, J=2.05 Hz, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH),3.77-3.90 (m, 1H, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 4.58-4.70 (m, 1H,—CH(CH₃)₂), 4.82-4.94 (m, 2H, —HNCH₂ -Pyr), 5.31-5.45 & 6.12-6.34 (m,1H, —NHCH(CH₂CH₃)CH(C(CH₃)₃)OH), 7.26-7.40 (m, 3H, 2×Pyr-H & —N═CH—N—),7.55 (s, 1H, Pyr-H), 7.75 (d, 1H, J=7.42 Hz, Pyr-H), 8.46-8.81 (m, 1H,HNCH₂-Pyr). FABMS m/z (relative intensity): 412 ([M+H]⁺, 100), 324 (70).Accurate Mass (M+H): Actual: 412.2825, Measured: 412.2835.

(3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (20 mg, 1eq, 0.07 mmol) in n-BuOH/DMSO (1.25 mL, 4:1) at room temperature underan argon atmosphere was added DIEA (0.25 mL, 20.5 eq, 1.44 mmol)followed by (3R)-3-amino-2-methyl-pentan-2-ol (22 mg, 2.7 eq, 0.19mmol). The reaction mixture was placed in a preheated oil bath at 140°C. and stirred at this temperature for 72 h. The reaction mixture wasallowed to cool to room temperature and the solvent was evaporated invacuo. The residue was partitioned between EtOAc (50 mL) and brine/water(1:1, 100 mL), the aqueous phase was extracted with more EtOAc (2×25mL), and the combined organic phase was washed with brine (50 mL), dried(MgSO₄) and evaporated in vacuo. The residue was purified by gradientcolumn chromatography on silica gel eluted with CHCl₃:MeOH (100:0→98:2),to afford the title compound as a white solid. Yield: 3.7 mg (14%).¹H-NMR (d-CDCl₃, 250 MHz): δ1.00 (t, 3H, J=7.27 Hz, —NHCH(CH₂ CH₃)C(CH₃)₂OH), 1.21 & 1.31 (2×s, 6H, —NHCH(CH₂CH₃)C(CH₃ ) ² OH), 1.57 (d,6H, J=6.63 Hz, —CH(CH₃ ) ² ), 1.69-1.89 (m, 2H, —NHCH(CH₂CH₃)C(CH₃)₂OH), 3.66-3.83 (m, 1H, —NHCH(CH₂CH₃)C(CH₃)₂OH), 4.59-4.73 (m,1H, —CH(CH₃)₂), 4.77-5.00 (m, 2H, —HNCH₂ -Pyr), 7.26-7.46 (m, 3H,2×Pyr-H & —N═CH—N—), 7.49-7.69 (m, 1H, Pyr-H), 7.78 (d, 1H, J=7.10 Hz,Pyr-H). FABMS m/z (relative intensity): 384 ([M+H]⁺, 65), 366 (23), 324(100), 217 (35), 192 (55). Accurate Mass (M+H): Actual: 384.2512,Measured: 384.2494.

(3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol

To a stirred solution of(2-fluoro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (30 mg, 1eq, 0.10 mmol) in n-BuOH/DMSO (1.25 mL, 4:1) at room temperature underan argon atmosphere was added DIEA (0.25 mL, 14.4 eq, 1.44 mmol)followed by (3S)-3-amino-2-methyl-pentan-2-ol (40 mg, 3.2 eq, 34 mmol).The reaction mixture was placed in a preheated oil bath at 140° C. andstirred at this temperature for 72 h. The reaction mixture was allowedto cool to room temperature and the solvent was evaporated in vacuo. Theresidue was partitioned between EtOAc (50 mL) and brine/water (1:1, 100mL), the aqueous phase was extracted with more EtOAc (2×25 mL), and thecombined organic phase was washed with brine (50 mL), dried (MgSO₄) andevaporated in vacuo. The residue was purified by gradient columnchromatography on silica gel eluted with CHCl₃:MeOH (100:0→98:2), toafford the title compound as a white solid. Yield: 6.5 mg (16%). ¹H-NMR(d-CDCl₃, 250 MHz): 1.00 (t, 3H, J=7.42 Hz, —NHCH(CH₂ CH₃ )C(CH₃)₂OH),1.22 & 1.31 (2×s, 6H, —NHCH(CH₂CH₃)C(CH₃ ) ² OH), 1.57 (d, 6H, J=6.79Hz, —CH(CH₃ ) ² ), 1.70-1.90 (m, 2H, —NHCH(CH₂ CH₃)C(CH₃)₂OH), 3.66-3.81(m, 1H, —NHCH(CH₂CH₃)C(CH₃)₂OH), 4.56-4.73 (m, 1H, —CH(CH₃)₂), 4.78-5.01(m, 2H, —HNCH₂ -Pyr), 7.20-7.45 (m, 3H, 2×Pyr-H & —N═CH—N—), 7.48-7.69(m, 1H, Pyr-H), 7.77 (d, 1H, J=7.74 Hz, Pyr-H). FABMS m/z (relativeintensity): 384 ([M+H]⁺, 100), 366 (30), 324 (85), 286 (40), 242 (65),192 (63), 176 (70). Accurate Mass (M+H): Actual: 384.2512, Measured:384.2494.

For the large-scale preparation of the individual enantiomers of3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol24 (Scheme 3 below), a different procedure was adopted. Dichloropurine14 was aminated at C-6 with pyridylmethylamine 15 to afford intermediate16, which was then alkylated at N-9 with 2-bromopropane 17. Theresulting chloropurine 18 was aminated at C-2 with the appropriateenantiomer of 3-amino-2-methyl-pentan-2-ol 23.

It has previously been demonstrated that optically pureR-3-amino-2-methyl-pentan-2-ol 23 can be obtained directly by conversionof R-2-amino-butyric acid 19 to the methyl ester 20, followed by doubleGrignard methylation (Guenther, B. R.; Kirmse, W. Liebigs Ann. Chem.,1980, 518). This method was not suitable for scale-up work in terms ofyield and product purity. Protection of the amino group as the dibenzylderivative (21, Bn=benzyl) was therefore adopted. Methylation of aminoester 21 provided alcohol 22 in high yield and purity. The benzyl groupswere then removed hydrogenolytically to afford the pure enantiomers of3-amino-2-methyl-pentan-2-ol 23.

(2-Chloro-9H-purin-6-yl)-pyridin-3-ylmethyl-amine

2,6-Dichloro-9H-purine (157.2 g, 0.83 mol), butan-1-ol (1.2 L), and Et₃N(290 mL, 2.08 mol) were stirred and heated to 50° C.3-C-Pyridin-3-yl-methylamine (94 mL, 0.76 mol) was added and thereaction heated to 120° C. for 1 h. Heating was ceased and the flaskallowed to cool to ambient temperature then to 0° C. using an ice-waterbath. The peach-coloured precipitate was collected by filtration, washedwith H₂O (1 L), hexane (2×0.5 L) and dried to leave a peach-colouredsolid. A second crop was obtained by evaporation of the combinedfiltrate and washings, trituration with H₂O (100 mL) and CHCl₃ (100 ml),filtration, washing with hexane, and drying. The crops were combined toprovide the title compound (196 g, 91%). ¹H-NMR (DMSO-d₆): δ 4.67-4.69(2H, d, —CH ₂—NH—), 5.20 (1H, br. s, NH), 7.34-7.39 (dd, ArH), 7.76-7.79(1H, d, ArH), 8.17 (1H, s, ArH —N═CH—N—), 8.46-8.48 (1H, d, ArH), 8.61(1H, s, ArH), 8.80 (1H, br. s, NH).

(2-Chloro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine

(2-Chloro-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (110.3 g, 0.37 mol)was dissolved in dry DMF (1.5 L) and warmed to 70° C. with stirring.K₂CO₃ (230 g, 1.67 mol) and 2-bromopropane (300 mL, 3.20 mol) were addedand the reaction was stirred at 70° C. overnight. The reaction mixturewas allowed to cool to room temperature and K₂CO₃ was removed byfiltration and washing with EtOAc. The combined filtrate and washingswere concentrated on a rotary evaporator and then under high vacuum toremove most of the DMF. The resulting residue was stirred vigorously inH₂O (1 L) and CH₂Cl₂ (1 L) until dissolution was complete. The aqueouslayer was extracted with more CH₂Cl₂ (2×200 mL), the organic extractswere combined, dried over MgSO₄, and evaporated to afford the titlecompound (87.2 g, 78%) as a beige powder. ¹H-NMR (DMSO-d₆): δ1.50-1.53(6H, d, —CH(CH ₃)₂), 4.67-4.72 (3H, m, —CH ₂—NH— & —CH(CH₃)₂), 5.20 (1H,br. s, NH, 7.34-7.39 (dd, ArH), 7.76-7.79 (1H, d, ArH), 8.33 (1H, s,ArH, —N═CH—N—), 8.46-8.48 (1H, d, ArH), 8.61 (1H, s, ArH), 8.92 (1H, br.s, NH.

R-2-Amino-butyric acid methyl ester hydrochloride

(D)-(−)-2-Aminobutyric acid (50.0 g, 0.48 mol) was suspended in MeOH(270 mL) and cooled to −10° C. (ice-salt bath) with stirring, under N₂.SOCl₂ (64 mL, 0.864 mol) was added dropwise, over 90 min. The flask wasfitted with a reflux condenser and heated to 70° C. for 1 h then cooledand evaporated (co-evaporating with MeOH). The residue was broken up anddried under high vacuum to afford the title compound as a white powder(75 g, 100%). ¹H-NMR (CDCl₃): δ0.88-0.95 (3H, t, CH ₃CH₂—), 1.48 (2H,br. s, NH₂), 1.50-1.80 (2H, m, CH₃CH ₂—), 3.35-3.40 (1H, t, CHNH₂), 3.69(3H, s, OCH).

R-2-Dibenzylamino-butyric acid methyl ester

To R-2-amino-butyric acid methyl ester hydrochloride (74.5 g, 0.485 mol)in dry DMF (500 mL) under N₂ at room temperature was added K₂CO₃ (147 g,1.065 mol), followed by dropwise addition of benzyl bromide (127 mL,1.065 mol.). After 2 h stirring K₂CO₃ was filtered off and washed(EtOAc, 200 mL). The combined filtrate and washings were evaporated invacuo. The oily residue was partitioned between EtOAc (1.5 L) and H₂O(1.5 L). The organic layer was separated and the aqueous layer wasextracted with more EtOAc (1 L). The combined organic fractions wereconcentrated. The residue was purified by dry silica gel flashchromatography (3:7 CH₂C₁₋₂/hexane). Pure fractions were combined,concentrated in vacuo, and dried to afford the title compound (136.1 g,94%). 1H-NMR (CDCl₃): δ0.90-0.96 (3H, t, CH ₃CH₂—), 1.75-1.81 (2H, m,CH₃—CH ₂—), 3.23-3.29 (1H, t, —CH₂—CH—), 3.50-3.57 (2H, d, —NCH ₂Ph),3.77 (3H, s, —OCH ₃), 3.93-3.99 (2H, d, —NCH ₂Ph), 7.22-7.41 (10H, m,ArH).

R-3-Dibenzylamino-2-methyl-pentan-2-ol

To a dried 5-L 3-necked flask fitted with a condenser and N₂ bubblerwere charged Mg turnings (53.7 g, 2.22 mol) and dry Et₂O (2 L). Themixture was stirred and cooled to 0° C. using an ice bath. MeI (138.2mL, 2.22 mol) was then added dropwise, over 1 h.R-2-Dibenzylamino-butyric acid methyl ester in dry Et₂O (1 L) was thenadded slowly (over 10 min) and the reaction was allowed to warm to roomtemperature, becoming very thick. The reaction was left stirring for 72h, cooled to 0° C. and quenched by slow addition of saturated aq NH₄Clsolution (350 g in 1 L). After 30 min stirring, the reaction mixture wasfiltered through a pad of Celite and washed with Et₂O (3×0.5 L). Theethereal layer was separated and the aqueous layer extracted with moreEt₂O (1 L). The organic fractions were combined, dried over MgSO₄ andconcentrated The residue was purified by silica gel flash chromatography(2% EtOAc in hexane) to afford the title compound (88.4 g, 82%) as acolourless oil. ¹H-NMR (CDCl₃): δ1.06 (3H, s, —CCH ₃), 1.15-1.26 (6H, m(3H s & 3H m, —CCH ₃ & —CH₂CH ₃), 1.50-1.70 (1H, m, —CHHCH₃), 1.70-1.90(1H, m, —CHHCH₃), 2.59-2.64 (1H, dd, —CHN—), 3.61-3.67 (2H, d, —NCH₂Ph), 3.97-4.02 (2H, d, —NCH ₂Ph), 4.25 (1H, br. s, —OH), 7.24-7.37(10H, m, ArH.

R-3-Amino-2-methyl-pentan-2-ol

R-3-Dibenzylamino-2-methyl-pentan-2-ol (21.0 g, 70.9 mmol) and Pd(C)(2.1 g, 20% Pd by dry weight, 50% H₂O) in MeOH (100 mL) were shaken in aParr hydrogenator at 65 psi overnight. The catalyst was removed byfiltration through a pad of Celite, the filtrate concentrated and driedunder high vacuum to afford the title compound as a clear yellow oil(7.585 g, 91%). ¹H-NMR (CDCl₃): δ0.90-1.05 (6H, m (3H s & 3H m), —CCH ₃& —CH₂CH ₃), 1.18 (3H, s, —CCH ₃), 1.55-1.62 (2H, m, —CH ₂CH₃),1.88-2.25 (2H, br. s, —NH ₂), 2.35-2.40 (1H, dd, —CHN—).

(3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol

(2-Chloro-9-isopropyl-9H-purin-6-yl)-pyridin-3-ylmethyl-amine (24.4 g,82.4 mmol), R-3-amino-2-methyl-pentan-2-ol (24.4 g, 209 mmol), and K₂CO₃(24.4 g, 177 mmol) were heated to 150° C. for 72 h under N₂. The mixturewas cooled, diluted with CH₂Cl₂, and K₂CO₃ removed by filtration. Thefiltrate was evaporated, then distilled (Kugelrohr) under high vacuum toremove unreacted R-3-amino-2-methyl-pentan-2-ol. The residue wassubjected to two rounds of silica gel flash chromatography (first columneluted with 5 to 10% linear gradient of MeOH in CH₂Cl₂; second columneluted with 2.5% MeOH in CH₂Cl₂). The residue was dissolved in hot EtOAc(150 mL), the solution concentrated to a volume of approximately 100 mLand allowed to cool. Et₂O (10 mL) was added to induce crystallizationand the solution was cooled to 0° C. The crystals were collected byfiltration, washed with a little Et₂O and dried to afford pure titlecompound (5.70 g, 18%) as an off-white solid. The combined filtrate andwashings were concentrated, the residue redissolved in the minimumvolume of hot Me₂CO and allowed to cool. The solid product was filteredand redissolved in the minimum volume of hot EtOAc and allowed to cool.The solids formed were removed by filtration and the filtrate was againconcentrated to dryness, redissolved in a mixture of hot Me₂CO andEtOAc, cooled, filtered and dried to afford a second crop of pure titlecompound (2.15 g, 5.6 mmol, 7%) as an off-white solid. M.p. 138-145° C.[α]_(D) ²⁰+32.54 (c 0.48, MeOH). LC-MS: 97.86% pure, m/z 384 [M+H]⁺, 406[M+Na]⁺, C₂₀H₂₉N₇O=383.49. Microanalysis (% found (theory)): C, 62.41(62.64); H, 7.56 (7.62); N, 25.76 (25.57). ¹H-NMR (CDCl₃): δ0.93-0.99(3H, t, —CH₂CH ₃), 1.17 & 1.27 (6H, 2s, —C(CH ₃)₂OH), 1.51-1.54 (6H, d,—NCH(CH₃)₂), 1.65-1.80 (2H, m, —CH ₂CH₃), 2.15 (1H, br. s, NH or OH),3.60-3.67 (m, 1H, —NHCH(CH₂CH₃)—), 4.57-4.62 (1H, m, —NCH(CH₃)₂),4.77-4.80 (2H, m, —HNCH ₂-Pyr), 5.15 (br. s, 1H, NH or OH), 6.14 (1H,br. s, NH or OH), 7.21-7.26 (1H, m, Pry-H), 7.48 (1H, s, —N═CH—N—),7.68-7.71 (app. d, 1H, Pyr-H), 8.51-8.52 (app. d, 1H, Pyr-H), 8.64 (1H,app. s, Pyr-H). ¹³C-NMR (CDCl₃): consistent with structure containing 20C atoms; 10 aliphatic C at δ10.21, 20.88, 20.92, 21.90, 23.09, 26.64,40.29 (weak), 44.72, 61.70 & 72.45 ppm; and 10 aromatic C at 112.96,121.73, 132.97, 133.07, 133.65, 146.98, 147.67, 148.89 (weak), 152.97 &158.77 ppm.

(3S)-3-{(9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol

This compound was obtained using procedures analogous to those describedabove for the (R) enantiomer but starting with (L)-(+)-2-aminobutyricacid. M.p. 140.5-146° C. [α]_(D) ²⁰−29.00 (c 0.46, MeOH). LC-MS: 97.85%pure, m/z 384 [M+H]⁺, C₂₀H₂₉N₇O=383.49. Microanalysis (% found(theory)): C, 62.51; (62.64); H, 7.61; (7.62); N, 25.70; (25.57).¹H-NMR: δ0.93-0.99 (3H, t, —CH₂CH ₃), 1.17 & 1.27 (6H, 2s, —C(CH ₃)₂OH),1.51-1.54 (6H, d, —NCH(CH₃)₂), 1.65-1.80 (2H, m, —CH ₂CH₃), 2.15 (1H,br. s, NH or OH), 3.60-3.67 (m, 1H, —NHCH(CH₂CH₃)—), 4.57-4.62 (1H, m,—NCH(CH₃)₂), 4.77-4.80 (2H, m, —HNCH ₂-Pyr), 5.15 (br. s, 1H, NH or OH),6.14 (1H, br. s, NH or OH), 7.21-7.26 (1H, m, Pry-H), 7.48 (1H, s,—N═CH—N—), 7.68-7.71 (app. d, 1H, Pyr-H), 8.51-8.52 (app. d, 1H, Pyr-H),8.64 (1H, app. s, Pyr-H). ¹³C-NMR (CDCl₃): consistent with structurecontaining 20 C atoms; 10 aliphatic C at δ10.21, 20.88, 20.92, 21.90,23.09, 26.64, 40.27 (weak), 44.71, 61.70 & 72.45 ppm; and 10 aromatic Cat 112.95, 121.72, 132.96, 133.07, 133.65, 146.85, 146.98, 148.86(weak), 152.97 & 158.77 ppm.

Various modifications and variations of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the relevant fields are intended to be covered by the presentinvention.

TABLE 1 Kinase inhibition (μM) CDK2/ CDK 1/ CDK4/ CDK7/ cyclin E cyclinB cyclin D1 cyclin H PKA ERK2 Name IC₅₀ SD IC₅₀ SD IC₅₀ SD IC₅₀ SD IC₅₀IC₅₀ SD Roscovitine 0.10 0.10 2.7 2.5 14 4 0.49 0.26 >50 1.2 1.3(2S3R)-3-{9-Isopropyl-6- 0.09 0.02 4.9 0.3 14 4 0.50 0.40 >200 23 12[(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-pentan-2-ol(2R3S)-3-{9-Isopropyl-6- 0.03 0.01 2.2 1.2 6.8 3.2 1.3 1.0 >200 20 6[(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-pentan-2-ol(3RS,4R)-4-{9-Isopropyl-6- 0.22 0.10 12 1 25 0 1.1 0.3 >200 37 6[(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-hexan-3-ol(3RS,4S)-4-{9-Isopropyl-6- 0.11 0.04 13 4 11 1 1.5 0.6 >200 17 8[(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-hexan-3-ol(3RS,4R)-4-{9-Isopropyl-6- 0.69 0.30 20 8 21 15 2.2 1.4 >200 111 11[(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-2-methyl- hexan-3-ol(3RS,4S)-4-{9-Isopropyl-6- 1.1 0.6 22 12 31 9 3.5 0.4 >200 67 6[(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-2-methyl- hexan-3-ol(3RS,4R)-4-{9-Isopropyl-6- 1.0 0.0 23 8 24 3 1.6 0.1 >200 51 8[(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol (3RS,4S)-4-{9-Isopropyl-6- 0.39 0.27 21 2 13 0 3.50.6 >200 23 4 [(pyridin-2-ylmethyl)-amino]- 9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol (3R)-3-{9-Isopropyl-6-[(pyridin- 0.20 0.08 6.2 1.17.5 5 3.1 0.4 >200 91 21 2-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol (3S)-3-{9-Isopropyl-6-[(pyridin- 0.07 0.014.4 1.9 12 1 2.5 1.5 >200 35 10 2-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol

TABLE 2 In vitro anti-proliferative activity (72-h MTT IC₅₀, μM) NameIC₅₀ ^(a) Stand. Dev. Roscovitine 12 3(2S3R)-3-{9-Isopropyl-6-[(pyridin- 1.5 0.43-ylmethyl)-amino]-9H-purin-2- ylamino}-pentan-2-ol(2R3S)-3-{9-Isopropyl-6-[(pyridin- 0.8 0.33-ylmethyl)-amino]-9H-purin-2- ylamino}-pentan-2-ol(3RS,4R)-4-{9-Isopropyl-6-[(pyridin- 10 1 3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-3-ol (3RS,4S)-4-{9-Isopropyl-6-[(pyridin- 5.5 2.83-ylmethyl)-amino]-9H-purin-2- ylamino}-hexan-3-ol(3RS,4R)-4-{9-Isopropyl-6-[(pyridin- 2.8 103-ylmethyl)-amino]-9H-purin-2- ylamino}-2-methyl-hexan-3-ol(3RS,4S)-4-{9-Isopropyl-6-[(pyridin- 19 2 3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-hexan-3-ol (3RS,4R)-4-{9-Isopropyl-6-[(pyridin- 19 23-ylmethyl)-amino]-9H-purin-2- ylamino}-2,2-dimethyl-hexan-3-ol(3RS,4S)-4-{9-Isopropyl-6-[(pyridin- 15 143-ylmethyl)-amino]-9H-purin-2- ylamino}-2,2-dimethyl-hexan-3-ol(3R)-3-{9-Isopropyl-6-[(pyridin- 5.6 2.1 3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol (3S)-3-{9-Isopropyl-6-[(pyridin- 4.0 0.53-ylmethyl)-amino]-9H-purin-2- ylamino}-2-methyl-pentan-2-ol ^(a)Humantumour cell lines: A549, HT29, Saos-2

TABLE 3 72-h MTT IC₅₀ (μM) (3S)-3-{9- (2S3R)-3-{9- (2R3S)-3-{9-Isopropyl-6- Isopropyl-6- Isopropyl-6- [(pyridin-3- [(pyridin-3-[(pyridin-3- ylmethyl)- ylmethyl)- ylmethyl)- amino]-9H- amino]-9H-amino]-9H- purin-3- purin-2- purin-2- ylamino}-2- Cell line ylamino}-ylamino}- methyl- Code Type Source Roscovitine pentan-2-ol pentan-2-olpentan-2-ol A549 Adherent Lung carcinoma  9.7 ± 1.1 2.1 ± 0.4 0.9 ± 0.13.6 ± 0.2 HeLa Adherent Cervical 16.5 ± 1.8 2.9 ± 0.2 1.4 ± 0.2 4.7 ±0.5 adenocarcinoma HT-29 Adherent Colorectal 10.2 ± 2.7 2.3 ± 0.6 1.1 ±0.2 2.3 ± 0.9 adenocarcinoma MCF7 Adherent Mammary  8.7 ± 0.5 1.6 ± 0.20.7 ± 0.1 2.3 ± 0.3 adenocarcinoma Saos-2 Adherent Osteosarcoma 15.3 ±4.2 2.8 ± 0.1 1.2 ± 0.2 3.9 ± 0.8 CCRF- Suspension Acute lymphoblastic16.4 ± 0.8 n.d. n.d. 3.7 ± 0.3 CEM leukaemia HL-60 Suspension Acutepromyelocytic 13.3 ± 1.1 n.d. n.d. 3.0 ± 0.2 leukaemia K-562 SuspensionChronic myelogenous 40.4 ± 4.4 n.d. n.d. 8.4 ± 1.2 leukaemia Mean(cancer cell lines)  16.3 ± 10.2 2.3 ± 0.5 1.1 ± 0.3 4.0 ± 2.0 Hs27Normal Foreskin fibroblast >20 10.2 ± 0.6  5.9 ± 0.3 11.3 ± 0.7  IMR90Normal Embryo lung >20 >20 >20 >20 fibroblast WI38 Normal Foetal lungfibroblast >20 11.2 ± 1.2  6.1 ± 0.3 >20 Mean (normal celllines) >20 >10 >10 >10

TABLE 4 A: % B: IC₅₀ C: IC₅₀ Compound CDK2 cytotox. % Drug remaining/roscovitine/ roscovitine/ after 30 min. % IC₅₀ IC₅₀ Clog microsomalroscovitine CDK2 cytotox. Name P incubation remaining compound compoundA × B A × C Roscovitine 3.7 33 1.0 1.0 1.0 1.0 1.0(2S3R)-3-{9-Isopropyl-6- 2.5 90 2.7 1.1 8.0 3.1 22[(pyridin-3-ylmethyl)- amino]-9H-purin-2- ylamino}-pentan-2-ol(2R3S)-3-{9-Isopropyl-6- 2.5 67 2.0 3.7 14 7.5 29 [(pyridin-3-ylmethyl)-amino]-9H-purin-2- ylamino}-pentan-2-ol (3RS,4R)-4-{9-Isopropyl- 3.0 702.1 0.5 1.1 1.0 2.4 6-[(pyridin-3-ylmethyl)- amino]-9H-purin-2-ylamino}-hexan-3-ol (3RS,4S)-4-{9-Isopropyl- 3.0 73 2.2 1.0 2.1 2.1 4.76-[(pyridin-3-ylmethyl)- amino]-9H-purin-2- ylamino}-hexan-3-ol(3RS,4S)-4-{9-Isopropyl- 3.8 50 1.5 0.3 0.8 0.4 1.26-[(pyridin-3-ylmethyl)- amino]-9H-purin-2- ylamino}-2,2-dimethyl-hexan-3-ol (3R)-3-{9-Isopropyl-6- 2.9 84 2.6 0.5 2.1 1.3 5.3[(pyridin-3-ylmethyl)- amino]-9H-purin-2- ylamino}-2-methyl- pentan-2-ol(3S)-3-{9-Isopropyl-6- 2.9 78 2.4 1.4 2.9 3.2 6.9 [(pyridin-3-ylmethyl)-amino]-9H-purin-3- ylamino}-2-methyl- pentan-2-ol

1. A compound of formula I

or a pharmaceutically acceptable salt thereof, wherein one of R¹ and R²is methyl, ethyl or isopropyl, and the other is H; R³ and R⁴ are eachindependently H, branched C₃-C₆ alkyl, or aryl, and wherein at least oneof R³ and R⁴ is other than H; R⁵ is a branched or unbranched C₁-C₅ alkylgroup or a C₃-C₆ cycloalkyl group, each of which may be optionallysubstituted with one or more OH groups; R⁶, R⁷, R⁸ and R⁹ are eachindependently H, halogen, NO₂, OH, OMethyl, CN, NH₂, COOH, CONH₂, orSO₂NH₂.
 2. A compound according to claim 1 wherein one of R¹ and R² isethyl or isopropyl, and the other is H.
 3. A compound according to claim1 wherein R⁵ is isopropyl or cyclopentyl.
 4. A compound according toclaim 1 wherein R⁶, R⁷, R⁸ and R⁹ are all H.
 5. A compound according toclaim 1 wherein one of R¹ and R² is ethyl and the other is H.
 6. Acompound according to claim 1 wherein R³ and R⁴ are each independentlyH, isopropyl, s-butyl, t-butyl or phenyl.
 7. A compound according toclaim 1 wherein R³ and R⁴ are each independently H, isopropyl, s-butylor t-butyl.
 8. A compound according to claim 1 wherein R³ and R⁴ areeach independently H, isopropyl or t-butyl.
 9. A compound according toclaim 1 selected from the following:(3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-hexan-3-ol;(3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl-hexan-3-ol;(3RS,4R)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol;and(3RS,4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2-dimethyl-hexan-3-ol.10. A pharmaceutical composition comprising a compound according toclaim 1 admixed with a pharmaceutically acceptable diluent, excipient orcarrier, or a mixture thereof.